Gas-fed fermentation reactors, systems and processes utilizing gas/liquid separation vessels

ABSTRACT

Reactors, systems and processes for the production of biomass by culturing microorganisms in aqueous liquid culture medium circulating inner loop reactor which utilize nonvertical pressure reduction zones are described. Recovery and processing of the culture microorganisms to obtain products, such as proteins or hydrocarbons is described.

BACKGROUND Technical Field

This invention is related to gas fed fermentation reactors, systems andprocesses useful in fermentation that utilize gas/liquid separationvessels and, in particular, fermentation systems using a gaseoussubstrate.

Description of the Related Art

With the ever increasing depletion of fossil fuel deposits, theincreasing production of greenhouse gases and recent concerns aboutclimate change, substituting biofuels (e.g., ethanol, biodiesel) forfossil fuels has become an industrial focus. However, biofuels generatedto date have their own difficulties and concerns. First generationbiofuels are derived from plants (e.g., starch; cane sugar; and corn,rapeseed, soybean, palm, and other vegetable oils), but these fuel cropscompete with crops grown for human and animal consumption. The amount ofglobally available farm land is insufficient to satisfy the increasingneeds for both food and fuel. To reduce the demand placed upon foodproducers for biofuel compatible grains, second generation biofuelsusing alternative biological material such as cellulose or algae areunder development. However, technical difficulties in production, alongwith the high cost of production, have not made second generationbiofuels any more cost-effective or accessible.

Third or next generation biofuels are made using alternative, non-foodbased, carbon feedstocks. As part of this effort, the use ofalternative, non-biological based, feedstocks, in the production ofhigher hydrocarbon compounds including fuels, lubricants, and plasticsis gaining ever-increasing momentum. Such feedstocks may include one ormore carbon-containing compounds or mixtures of carbon-containing andnon-carbon-containing compounds that include, among others, methane andsyngas. Methane, for example, is a relatively abundant, naturallyoccurring and found in many locations throughout the world. Methane isalso produced during many biological decay processes, and thus may becaptured from waste treatment and landfill facilities. For its relativeabundance, methane is a potent greenhouse gas, having 23× the relativegreenhouse gas contribution of CO₂. Historically, methane has beenviewed as a somewhat valuable byproduct that is difficult to convert tohigher value products or to transport to the marketplace from remote orstranded locations such as remote gas fields or off-shore productionplatforms. Methane from such sources, as well as the methane produced bybiological decomposition processes occurring at sewage treatmentfacilities and landfills, is primarily either vented or flared. Theability to economically and efficiently convert methane and similarcarbon-containing gases to one or more higher value C₂ or higherhydrocarbons would permit producers to take advantage of a relativelyabundant, non-biologically produced, feedstock while, at the same time,providing a significant environmental benefit.

The rise in domestic production of methane capability makes methane morereadily available domestically. Domestic natural gas is primarilyproduced by hydraulic fracturing (“fracking”), but methane can also beobtained from other sources, such as landfills and sewage. But methane'svolatility makes the transport and/or direct usage of methane as a fuelproblematic.

For these reasons, a strong incentive exists to convert the methane toone or more liquid products, for example motor fuels, to permit easiertransport to the point of use or sale. Two main approaches are currentlybeing pursued: liquefaction leading to liquefied natural gas (LNG) andchemical conversion to convert gas-to-liquid (GTL) (Patel, 2005, 7thWorld Congress of Chemical Engineering, Glasgow, Scotland, UK). TheFischer Tropsch (F-T) process is currently the most prevalent approachfor converting large quantities of methane to higher-order hydrocarbons(Patel, 2005). Note that the F-T process takes syngas as an input;syngas is produced from natural gas by steam reforming (syngas can alsobe sourced from coal gasification, by high temperature reaction withwater and oxygen). The F-T process yields petroleum products consistentwith today's fuel supply, but suffers from a number of drawbacks,including low yields, poor selectivity (making downstream utilizationcomplex), and requires significant capital expenditure and scale toachieve economical production (Spath and Dayton, December 2003NRELITP-510-34929). The massive scale required for an F-T plant(generally in excess of two billion dollars in capital cost [Patel,2005]) also represents a significant limitation due to the large amountof methane feedstock required to offset the enormous capital cost of theF-T process. As methane transportation is prohibitively expensive inmost cases, such a plant must be co-located with a steady, reliable, andcost efficient source of methane, usually in the form of a significantmethane reservoir or a methane pipeline. An additional cost and scalingfactor is the economics of gas-scrubbing technologies (Spath and Dayton,2003), since F-T catalysts are quite sensitive to common contaminantsfound in natural gas that pass unaffected through the syngas conversionprocess.

The requirements for ready access to large volumes of a relatively cleanmethane-containing gas, combined with a massive capital investment,currently limit natural gas based F-T plants to successful andeconomically viable operation in only a few locations worldwide (Spathand Dayton, 2003). The high minimum processing requirement for agas-to-liquids process or liquified natural gas plant, combined with thehigh cost of transport, result in smaller methane sources remaining as“stranded” gas deposits. Such stranded gas can include, but is notlimited to, natural gas produced at off-shore oil wells, or methaneoff-gas from landfills. Due to the current absence of efficientsmall-scale conversion technologies, such stranded gas sources aretypically vented to atmosphere or flared, as methane accumulationpresents a significant safety risk. Gas-to-liquids facilities using theFischer-Tropsch process have been in operation semi-continuously since1938. Several companies are currently investigating introduction of newplants given the current availability and price of methane discussedabove. However, despite significant research and development over thelast 70+ years, the limitations of Fischer-Tropsch technology preventbroad adoption of commercial gas-to-liquids processes.

Advances in the efficiency in animal feed utilization have been achievedover the past several decades through the use of feed additives. Theseadded substances augment the nutrient content, energy content, and/ordisease fighting properties of animal feed compositions. A growingchallenge for commercial animal producers is the rising cost of grain.The rising costs are due in part to competing demands for grains forbiofuel and human food use. With the rising cost of grain and proteincomplements, coupled with limited land available for feed production,alternative low-cost animal feed products with beneficial nutritive anddisease fighting properties are desirable.

A number of different protein-containing materials have been proposed assubstitutes for more traditional sources of protein, such as fish meal,soya products and blood plasma, in human foods and as animal feed. Theseprotein-containing materials include single cell microorganisms such asfungi, yeasts and bacteria which contain high proportions of proteins.These microorganisms may be grown on hydrocarbon or other substrates.

In view of the above, biological fermentation using C₁ substrates as acarbon source presents an attractive solution to both the currentcompetition between food sources and fermentation for producingchemicals/fuels, the need for alternative low-cost animal feed products,as well as the lack of good options for utilization of natural gas.However, fermentation of gaseous substrates such as methane, CO, or CO₂presents significant challenges due to the requirement that the carbonsubstrate must be transferred from the gas phase to an aqueous phase toallow for uptake and metabolism by the C₁ metabolizingnon-photosynthetic microorganisms in culture. Simultaneously, othergasses such as O₂ or H₂ may also be required to be transferred from thegas phase to allow cellular metabolism to progress (aerobic or anaerobicmetabolism, respectively). Waste products (such as CO₂ in the case ofaerobic metabolism) must be isolated from the microorganisms to allowfor efficient microbial growth. Further, the heat generation frommetabolism of C₁ substrates is significant and the system requirescooling to maintain optimal conditions for microbial growth. Inaddition, biological fermentation of C₁ substrates, sometimes results inC₁ substrates, such as methane, being in the same vessel as an oxidizingagent, such as oxygen. Care must be taken to avoid combustion anddeflagration.

Convective mass transfer from the liquid phase to the vapor phase can bedescribed with a mass transfer coefficient. The flux is equal to theproduct of the mass transfer coefficient, the surface area, and theconcentration difference (Flux=k A ΔC).

The mass transfer coefficient is influenced by a variety of factorsincluding the size of the molecule to be transferred, its solubility inthe aqueous phase, and the size of the boundary layer between the phases(typically controlled in fermentation systems by mixing speed andturbulence). The surface area between the gas and liquid phases in mostfermentation systems is primarily limited by the bubble size of theinput gas. Bubble size can be controlled by introducing the gas throughsmall pores, as well as increasing shear forces to break apart bubblesand prevent coalescence. The concentration difference can be theconcentration difference across the gas phase boundary layer, theconcentration difference across the liquid phase boundary layer, theconcentration difference between the bulk vapor and the vapor whichwould be in equilibrium with the bulk liquid, or the concentrationdifference between the bulk liquid and the liquid which would be inequilibrium with the bulk vapor. In most fermentation systems, theconcentration difference is controlled by the pressure of the gas phase.

Conventional fermentation systems (bioreactors) achieve gas mixing byone of two methods: stirring or airlift. Stirred fermentors achievemixing by means of stirring blades generally placed centrally in asingle large fermentor. The stirrer blades generate turbulence and shearin the liquid while gas bubbles are introduced at the bottom of thefermentor, thus impeding the progress of the bubbles as they travel upthe fermentor and shearing the gas bubbles to reduce the tendency of thebubbles to coalesce within the fermentor. The advantage of this type offermentor is the fast, relatively homogeneous mixing and gas bubbledispersion that is possible due to the high speed of the mixing blades.However, this type of fermentor can be difficult to scale-up, as theenergy requirements to obtain the same rate of mixing and mass transportcan be prohibitive as the volume increases. Further, the vigorous mixingimplies a significant heating of the fermentation liquid, and the use ofa single large fermentor limits the surface area available for heatexchange cooling.

Airlift fermentors avoid mechanical stirrers by incorporating a flowpath for the liquid. Airlift fermentors have a downflow and an upflowsection which are interconnected at both ends; these sections can eitherbe separate units (referred to as a loop fermentor), or concentric(airlift fermentor). In either case, gasses are supplied at the bottomof the upflow section through a bubble-generating apparatus. The bubblesmix with the liquid, reducing the density of the liquid and causing thegas-liquid mixture to rise through the upflow section. The risingmixture displaces liquid at the top of the reactor, which travels downthe downflow section to replace the liquid at the bottom, establishing acircular flow in the fermentor. In order to obtain a long residence timefor the gas bubbles in the liquid, airlift fermentors are generally talland have a limited transverse cross-sectional area. This implies thatthe gas must be supplied at a relatively high pressure to overcomehydrostatic pressure formed by the column of liquid present in thefermentor. In addition, the bubble size increases significantlythroughout the fermentor as the pressure decreases with height. Theincreasing bubble diameter proportionately reduces the rate of masstransfer between the gas bubbles and the liquid phase by reducing theratio of gas bubble area (proportionate to the square of the gas bubbleradius) to gas bubble volume (proportionate to the cube of the gasbubble radius) through which mass transfer may occur. Flow rates andshear forces in airlift fermentors are significantly lower than instirred tank fermentors, which also tend to increase bubble coalescenceand reduce the efficiency of cooling the fermentor. Finally, separationof the unused and waste gases from the mixture exiting the upflowportion of the fermentor prior to the return of the liquid to thedownflow section can be challenging.

Loop reactors are described in U.S. Pat. No. 7,575,163 and have beenproposed for fermenting microorganisms, e.g., for the generation ofbiomass or for the preparation of materials produced by microorganisms.FIG. 1 of U.S. Pat. No. 7,575,163 illustrates one loop reactor 1including an effluent gas removal zone 2 which flows into a verticaldownflow zone 3. Effluent gas removal zone 2 includes an outlet port 7and an emergency vent 8. Vertical downflow zone 3 includes a nutrientgas inlet 15. A propeller 10 powered by motor 11 assists in circulationof a liquid culture medium through the loop reactor. Upstream ofpropeller 10 is an exit port 12 for removing material from the loopreactor. Downstream of propeller 10 are ammonia and mineral inlets 17and 18. Liquid culture medium 9 passes through a plurality of staticmixers 14 in a horizontal section 4 of the loop reactor. The horizontalsection of the loop reactor also includes a plurality of nutrient gasinlets 13. Downstream of the last static mixer 14, the loop reactorincludes a vertical upflow section 5. The top end of vertical upflowsection 5 fluidly communicates with a substantially horizontal outflowzone 6. Vertical upflow section 5 is provided with a nutrient gas inlet16. Upstream of nutrient gas inlet 16 is a drive gas inlet 19 throughwhich a driving gas is delivered to the liquid culture medium. The '163patent describes the substantially horizontal outflow zone 6 isdesirable from the standpoint of making degassing of an effluentgas/liquid culture medium particularly effective.

BRIEF SUMMARY

In one aspect, the present disclosure describes systems, processes andapparatuses for efficient mass transfer of gaseous substrates formicrobial fermentation. Additionally, this disclosure describes systems,processes and apparatuses for fermenting gaseous carbon-containingfeedstocks using a culture primarily comprising a C₁ metabolizingnon-photosynthetic microorganism. In other aspects, this disclosuredescribes systems, processes and apparatuses for fermenting gaseousfeedstocks which include gaseous substrates, using other than C₁metabolizing non-photosynthetic microorganism(s). In yet another aspect,this disclosure describes scalable fermentor designs for allowing highflux gas-phase to liquid-phase mass transfer in addition to efficientgas/liquid separation and gas removal. Systems and processes forfermentation that overcome disadvantages known in the art and providethe public with new and safe processes and devices for the optimalproduction of a variety of products are described.

Such fermentation systems may employ one or more species ofmicroorganism that are capable of metabolizing gaseous compounds; forexample, C₁ compounds. Such microorganisms include prokaryotes orbacteria, such as Methylomonas, Methylobacter, Methylococcus,Methylosinus, Methylocystis, Methylomicrobium, Methanomonas,Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium,Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter,Rhodopseudomonas, or Pseudomonas. In some instances, the C₁ metabolizingmicroorganisms may include methanotrophs, methylotrophs or combinationsthereof. Preferred methanotrophs include Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, or combinations thereof. Exemplary methanotrophs includeMethylomonas sp. 16a (ATCC PTA 2402), Methylosinus trichosporium (NRRLB-II, 196), Methylosinus sporium (NRRL B-II, 197), Methylocystis parvus(NRRL B-II, 198), Methylomonas methanica (NRRL B-5 11,199), Methylomonasalb us (NRRL B-II, 200), Methylobacter capsulatus (NRRL B-11,201),Methylobacterium organophilum (ATCC 27,886), Methylomonas sp. AJ-3670(FERM P-2400), Methylomicrobium alcaliphilum, Methylocella silvestris,Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylosinustrichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas sp. 16a,Methylomicrobium alcaliphilum 20Z, or high growth variants thereof.Preferred methylotrophs include Methylobacterium extorquens,Methylobacterium radiotolerans, Methylobacterium populi,Methylobacterium chloromethanicum, Methylobacterium nodulans, orcombinations thereof.

Microorganisms capable of metabolizing C₁ compounds found in syngasinclude, but are not limited to Clostridium, Moorella, Pyrococcus,Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium,Acetobacterium, Acetoanaerobium, Butyribacterium, Peptostreptococcus, orcombinations thereof. Exemplary methylotrophs include Clostridiumautoethanogenum, Clostridium jungdahli, Clostridium ragsdalei,Clostridium carboxydivorans, Butyribacterium methylotrophicum,Clostridium woodii, Clostridium neopropanologen, or combinationsthereof. In some instances, C₁ metabolizing microorganisms areeukaryotes such as yeast, including Candida, Yarrowia, Hansenula,Pichia, Torulopsis, or Rhodotorula.

In other instances, the C₁ metabolizing non-photosynthetic microorganismis an obligate C₁ metabolizing non-photosynthetic microorganism, such asan obligate methanotroph, an obligate methylotroph, or combinationsthereof. In some instances, the C₁ metabolizing non-photosyntheticmicroorganism is a recombinant microorganism comprising a heterologouspolynucleotide encoding a fatty acid producing enzyme, a formaldehydeassimilation enzyme, or combinations thereof.

In addition to the above, the present disclosure describes the followingembodiments. A first embodiment directed to a system for stimulatingproduction of biomass that includes a loop reactor which includes afluid moving device having an inlet and an outlet with the inlet of thefluid moving device in fluid communication with an outlet of asubstantially vertical downflow zone. The loop reactor includes a loopsection including an outlet and an inlet, the inlet of the loop sectionin fluid communication with the outlet of the fluid moving device and asubstantially vertical upflow zone including an outlet and an inlet withthe inlet of the vertical upflow zone in fluid communication with theoutlet of the loop section. The loop reactor further includes agas/liquid separation vessel having a longitudinal axis and including anoutlet, an inlet wherein the inlet of the gas/liquid separation vesselis located in a lower portion of the gas/liquid separation vessel and influid communication with the outlet of the substantially vertical upflowzone. The outlet of the horizontal gas/liquid separation vessel is influid communication with the inlet of the substantially verticaldownflow zone. The gas/liquid separation vessel includes an intermediatesection between the outlet and the inlet of the gas/liquid separationvessel with the intermediate section having a constant diameter D, anoutlet side section on one side of the intermediate section, the outletside section including the outlet of the gas/liquid separation vesseland an inlet side section on a side of the intermediate section oppositethe outlet side section, the inlet side section including the inlet ofthe gas/liquid separation vessel and having a shape of an obliqueconical frustum with an increasing diameter in a direction of fluid flowthrough the inlet side section. The system also includes a fluid conduitof non-increasing diameter extending between the outlet of thegas/liquid separation vessel and the inlet of the fluid moving device.

A second embodiment disclosed herein is directed to the first embodimentfurther including a drain conduit including in inlet end and an outletend, the inlet end of the drain conduit connected to the outlet sidesection of the gas/liquid separation vessel and the outlet end of thedrain conduit connected to the substantially vertical downflow zone.

A third embodiment disclosed herein is directed to the system of thefirst and second embodiments, wherein a lowermost edge of the inlet, theoutlet, the intermediate section, the outlet side section and the inletside section of the gas/liquid separation vessel contact a common plane.

A fourth embodiment disclosed herein is directed to the first throughthird embodiments further comprising a feed conduit including an inletand an outlet, the inlet of the feed conduit connected to thesubstantially vertical upflow zone and the outlet end of the feedconduit connected to the inlet side section of the gas/liquid separationvessel.

A fifth embodiment disclosed herein is directed to the first throughfourth embodiments wherein the inlet side section has a shape of anoblique conical frustum which includes a cone angle that ranges between5 to 30°.

A sixth embodiment disclosed herein is directed to the first throughfifth embodiment wherein the outlet of the gas/liquid separation vesselhas a diameter that is less than a diameter of the inlet of thegas/liquid separation vessel.

A seventh embodiment disclosed herein is directed to the first throughsixth embodiments wherein the inlet side section has a length measuredalong the longitudinal axis of the gas/liquid separation vessel that isgreater than a length of the intermediate section measured along thelongitudinal axis of the gas/liquid separation vessel and greater than alength of the outlet side section measured along the longitudinal axisof the gas/liquid separation vessel.

An eighth embodiment disclosed herein is directed to the first throughseventh embodiments wherein the outlet side section has a dished shape.

A ninth embodiment disclosed herein is directed to a gas/liquidseparation vessel that includes an outlet, an inlet, and a longitudinalaxis, with the inlet of the gas/liquid separation vessel located in alower portion of the gas/liquid separation vessel. The gas/liquidseparation vessel includes an intermediate section between the outletand the inlet of the gas/liquid separation vessel with the intermediatesection having a constant diameter equal to D. An outlet side section isprovided on one side of the intermediate section, the outlet sidesection including the outlet of the gas/liquid separation vessel. Aninlet side section is provided on a side of the intermediate sectionopposite the outlet side section with the inlet side section includingthe inlet of the gas/liquid separation vessel and having a shape of anoblique conical frustum with an increasing diameter in a direction offluid flow through the inlet side section.

A tenth embodiment described herein is directed to the ninth embodimentwherein a lowermost edge of the inlet, the outlet, the intermediatesection, the outlet side section and the inlet side section of thegas/liquid separation vessel contact a common plane.

An eleventh embodiment described herein is directed to the ninth andtenth embodiments wherein the inlet side section has a shape of anoblique conical frustum includes a cone angle that ranges between 5 to30°.

A twelfth embodiment described herein is directed to the ninth througheleventh embodiments wherein the outlet of the gas/liquid separationvessel has a diameter that is less than a diameter of the inlet of thegas/liquid separation vessel.

A thirteenth embodiment described herein is directed to the ninththrough twelfth embodiments wherein the inlet side section has a lengthmeasured along the longitudinal axis of the gas/liquid separation vesselthat is greater than a length of the intermediate section measured alongthe longitudinal axis of the gas/liquid separation vessel and greaterthan a length of the outlet side section measured along the longitudinalaxis of the gas/liquid separation vessel.

A fourteenth embodiment described herein is directed to the ninththrough thirteenth embodiments, wherein the outlet side section has adished shape.

A fifteenth embodiment described herein is directed to a process forstimulating production of biomass that includes the steps of flowingthrough a loop section of a loop reactor, a multi-phase mixture of a gasand a liquid culture medium, introducing nutrients into the multi-phasemixture, introducing methane and oxygen into the multi-phase mixture,separating the multi-phase mixture of a gas and a liquid culture mediuminto a gas phase and a liquid phase in a gas/liquid separation vessel.Separating the multi-phase mixture into a gas phase and a liquid phasein the gas/liquid separation vessel including the steps of flowing themulti-phase mixture of a gas and a liquid culture medium into a lowerportion of the gas/liquid separation vessel through an inlet sidesection of the gas/liquid separation vessel wherein the inlet sidesection of the gas/liquid separation vessel has a shape of an obliqueconical frustum and an increasing diameter in a direction of flow of themulti-phase mixture through the inlet side section; flowing themulti-phase mixture through an intermediate section located between anoutlet side section of the gas/liquid separation vessel and the inletside section of the gas/liquid separation vessel with the intermediatesection having a constant diameter; and removing the liquid phase froman outlet of the gas/liquid separation vessel and delivering the removedliquid phase to an inlet of the loop section.

A sixteenth embodiment described herein is directed to the fifteenthembodiment wherein delivering the removed liquid phase to the inlet ofthe loop section includes flowing the removed liquid phase through aconduit of non-increasing diameter.

A seventeenth embodiment described herein is directed to the sixteenthembodiment wherein separating the multi-phase mixture of a gas and aliquid culture medium into a gas phase and a liquid phase in thegas/liquid separation vessel further includes flowing the multi-phasemixture through the inlet side section and the intermediate section ofthe gas/liquid separation vessel, the inlet side section and theintermediate section including a lowermost edge that contacts a commonplane.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the drawings, the sizes and relative positions of elements in thedrawings are not necessarily drawn to scale. For example, the variouselements and angles are not drawn to scale, and some of these elementsare arbitrarily enlarged and positioned to improve drawing legibility.Further, the particular shapes of the elements as drawn are not intendedto convey any information regarding the actual shape of the particularelements, and have been selected solely for ease of recognition in thedrawings.

FIG. 1 shows a schematic view of a system for stimulating production ofbiomass according to one or more described embodiments.

FIG. 2 shows a schematic block diagram of subsystems of a system forstimulating production of biomass according to one or more describedembodiments.

FIG. 3 shows a perspective view of an example of a gas/liquid separationvessel for use in a system for stimulating production of biomassaccording to one or more illustrated and described embodiments.

FIG. 4 shows an elevational view of a side of the gas/liquid separationvessel shown in FIG. 3.

FIG. 5 shows a top view of the gas/liquid separation vessel shown inFIG. 3.

FIG. 6 shows a bottom view of the gas/liquid separation vessel shown inFIG. 3.

FIG. 7 shows an elevational view from the left or downstream end of thegas/liquid separation vessel shown in FIG. 3.

FIG. 8 shows an elevational view from the right or upstream end of thegas/liquid separation vessel shown in FIG. 3.

FIG. 9A shows a vertical cross-section through a gas/liquid separationvessel shown in FIG. 4 and taken along line 9A-9A in FIG. 4.

FIG. 9B shows a vertical cross-section through a gas/liquid separationvessel shown in FIG. 4 and taken along line 9B-9B in FIG. 4.

FIG. 10 shows a high level flow diagram of a fermentation process thatincludes flowing a multi-phase mixture through a loop reactor accordingto one or more illustrated and/or described embodiments.

FIG. 11 shows a top view of another example of a gas/liquid separationvessel for use in a system for stimulating production of biomassaccording to one or more illustrated and described embodiments.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, structures,standard vessel design details, detailed design parameters of availablecomponents such as liquid or gas distributors, pumps, turbines, andsimilar, details concerning the design and construction of AmericanSociety of Mechanical Engineers (ASME) pressure vessels, control systemtheory, specific steps in one or more fermentation processes, and thelike have not been shown or described in detail to avoid unnecessarilyobscuring descriptions of the described embodiments. Unless the contextrequires otherwise, throughout the specification and claims whichfollow, the word “comprise” and variations thereof, such as, “comprises”and “comprising” are to be construed in an open, inclusive sense, thatis, as “including, but not limited to.” Further, headings providedherein are for convenience only and do not interpret the scope ormeaning of the claimed invention.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. Also, as used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the content clearly dictatesotherwise. It should also be noted that the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

Fermentors are generally defined as any vessel in which a fermentationprocess is carried out. Given the vast number of fermentation processesand the wide variety of fermentable substrates, fermentors can rangefrom simple continuous stirred tank reactors found in the alcoholicbeverage industry to highly complex, specialized vessels having gasdistribution and internal structures tailored to a particular substrateand/or a particular biological species. Fermentors useful in convertingcarbon-containing gases such as methane and syngas (a mixture of CO andH₂) to longer chain gaseous and liquid hydrocarbons generally disperse agas substrate containing the C₁ carbon compound within a liquid mediacontaining one or more nutrients to provide a multi-phase mixture. Thismulti-phase mixture is fed to one or more microbiological colonies thatconvert a portion of the C₁ carbon compound(s) in the gas substrate tomore preferred, longer chain, C₂ or higher compounds. The substratecomposition, nutrients, and microbiological organisms comprising thecolony (i.e., the biomass within the fermentor) can be variouslyadjusted or tailored to provide a desired final matrix of C₂ or highercompounds which may be present as a liquid, gas, or intracellularmaterial.

Fermentors useful in utilizing carbon-containing gases such as methaneand syngas (a mixture of CO and H₂) as a substrate for culturing singlecell microorganisms such as fungi, yeasts and bacteria which containhigh proportions of proteins generally disperse a gas substratecontaining a C₁ carbon compound within a liquid media containing one ormore nutrients to provide a multi-phase mixture. This multi-phasemixture is contacted with one or more microbiological colonies thatconvert a portion of the C₁ carbon compound(s) in the gas substrate toproteins. The substrate composition, nutrients, and microbiologicalorganisms comprising the colony (i.e., the biomass within the fermentor)can be variously adjusted or tailored to provide a desired final matrixof protein-containing biomass.

From a mass transfer perspective, gas substrate fermentors present aunique challenge in that the substrate is trapped within a gas bubbleand in order for microbiological uptake of the substrate to occur, thegas substrate must first pass from the gas bubble to the microbiologicalorganisms either directly or indirectly via dissolution in the liquidmedia. Such fermentation processes are thus frequently limited by theability of the system to facilitate and/or sustain a desirably highlevel of mass transfer of the substrate from the gas bubbles to themicrobiological organisms within the fermentor. At the least, the rateof mass transfer from the gas bubble to either the surrounding liquidmedia or to a microbiological organisms is a function of the gaspressure within the gas bubble, the volume to surface area ratio of thegas bubble, and the contact time of the gas bubble with the surroundingliquid or microbiological organisms. Increasing the pressure within thegas bubble or increasing the contact time of the gas bubble with thesurrounding liquid or microbiological organisms results in a highereffective mass transfer rate between the substrate and themicrobiological organisms. Decreasing the volume to surface area ratioof the gas bubble (i.e., reducing the diameter of the gas bubbles)results in a higher effective mass transfer rate between the gas bubbleand the surrounding liquid. Preferred fermentors from a mass transferstandpoint would therefore generate a large number of relatively smalldiameter gas bubbles at a relatively high pressure that are held inclose or intimate contact with the surrounding liquid or microbiologicalorganisms for an extended period of time.

Disclosed herein are a number of fermentation systems, methods, andapparatuses that are capable of providing relatively small diameter,relatively high pressure gas bubbles. Disclosed herein are a number offermentation systems, methods, and apparatuses capable of providing anextended contact time with the surrounding liquid and/or biologicalorganism(s). Such fermentation systems, methods, and apparatuses canadvantageously provide a highly efficient gas substrate fermentationsystem that may be particularly useful in converting C₁ compounds tomore preferred gaseous, liquid, and intra-cellular C₂ and highercompounds or stimulating the growth of microorganisms containing highproportions of protein.

As used herein, the terms “C₁ substrate” or “C₁ compound” refer to anycarbon-containing molecule or composition that lacks a carbon-carbonbond. Sample C₁ molecules or compositions include methane, methanol,formaldehyde, formic acid or a salt thereof, carbon monoxide, carbondioxide, syngas, methylamines (e.g., monomethylamine, dimethylamine,trimethylamine), methylthiols, or methylhalogens.

As used herein, the term “microorganism” refers to any microorganismhaving the ability to use a gaseous substrate as a source of energy oras its sole source of energy and biomass, and may or may not use othercarbon substrates (such as sugars and complex carbohydrates) for energyand biomass. Examples of microorganisms as used herein include theheterotrophic bacteria Ralstonia sp. (formerly Alcaligenes acidovorans)DB3 (strain NCIMB 13287), Brevibacillus agri (formerly Bacillus firmus)DB5 (strain NCIMB 13289) and Aneurinibacillus sp. (formerly Bacillusbrevis) DB4 (strain NCIMB 13288) which each have optimum growth at atemperature of about 45° C. Ralstonia sp. DB3 is a gram-negative,aerobic, motile rod belonging to the family Pseudomonadaceae which canuse ethanol, acetate, propionate and butyrate for growth.Aneurinibacillus sp. DB4 is a gram-negative, endospore-forming, aerobicrod belonging to the genus Bacillus which can utilize acetate,D-fructose, D-mannose, ribose and D-tagatose. Brevibacillus agri DB5 isa gram-negative, endospore-forming, motile, aerobic rod of the genusBacillus which can utilize acetate, N-acetyl-glucosamine, citrate,gluconate, D-glucose, glycerol and mannitol. Suitable yeasts for use inthe processes of the invention may be selected from the group consistingof Saccharomyces and Candida.

If desired, the processes described herein may be performed usingbacteria (or yeasts) genetically modified so as to generate a desiredchemical compound which can then be extracted from the intercellularfluid or the biomass harvested from the reactor. The scientific andpatent literature contains numerous examples of such geneticallymodified microorganisms including, inter alia, methanotrophic bacteria.

In at least some instances in accordance with embodiments describedherein, the microbiological organisms used to ferment gaseouscarbon-containing feedstocks employ a culture primarily comprising a C₁metabolizing non-photosynthetic microorganism. Such fermentation systemsmay use one or more species of C₁ metabolizing microorganisms that areprokaryotes or bacteria, such as Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, Methylophilus, Methylobacillus, Methylobacterium,Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia,Arthrobacter, Rhodopseudomonas, or Pseudomonas. In some instances, theC₁ metabolizing bacteria may include a methanotroph or a methylotroph.Preferred methanotrophs include Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium,Methanomonas, or a combination thereof. Exemplary methanotrophs includeMethylomonas sp. 16a (ATCC PTA 2402), Methylosinus trichosporium (NRRLB-II, 196), Methylosinus sporium (NRRL B-II, 197), Methylocystis parvus(NRRL B-II, 198), Methylomonas methanica (NRRL B-5 11,199), Methylomonasalb us (NRRL B-II, 200), Methylobacter capsulatus (NRRL B-11,201),Methylobacterium organophilum (ATCC 27,886), Methylomonas sp. AJ-3670(FERM P-2400), Methylomicrobium alcaliphilum, Methylocella silvestris,Methylacidiphilum infernorum, Methylibium petroleiphilum, Methylosinustrichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas sp. 16a,Methylomicrobium alcaliphilum 20Z, or a high growth variants thereof.Preferred methylotrophs include Methylobacterium extorquens,Methylobacterium radiotolerans, Methylobacterium populi,Methylobacterium chloromethanicum, Methylobacterium nodulans, or acombination thereof.

Microorganisms capable of metabolizing C₁ compounds found in syngasinclude, but are not limited to Clostridium, Moorella, Pyrococcus,Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium,Acetobacterium, Acetoanaerobium, Butyribacterium, Peptostreptococcus, orcombinations thereof may also be used. Exemplary methylotrophs includeClostridium autoethanogenum, Clostridium ljungdahli, Clostridiumragsdalei, Clostridium carboxydivorans, Butyribacteriummethylotrophicum, Clostridium woodii, Clostridium neopropanologen, or acombination thereof. In some instances, C₁ metabolizing microorganismsare eukaryotes such as yeast, including Candida, Yarrowia, Hansenula,Pichia, Torulopsis, or Rhodotorula.

In other instances, the C₁ metabolizing non-photosynthetic microorganismis an obligate C₁ metabolizing non-photosynthetic microorganism, such asan obligate methanotroph or methylotroph. In some instances, the C₁metabolizing non-photosynthetic microorganism is a recombinantmicroorganism comprising a heterologous polynucleotide encoding a fattyacid producing enzyme, a formaldehyde assimilation enzyme, or acombination thereof.

As used herein, the terms “C₁ metabolizing microorganism” or “C₁metabolizing non-photosynthetic microorganism” refer to anymicroorganism having the ability to use a single carbon (C₁) substrateas a source of energy or as its sole source of energy and biomass, andmay or may not use other carbon substrates (such as sugars and complexcarbohydrates) for energy and biomass. For example, a C₁ metabolizingmicroorganism may oxidize a C₁ substrate, such as methane or methanol.C₁ metabolizing microorganisms include bacteria (such as Methanotrophsand Methylotrophs) and yeast. In at least some instances, a C₁metabolizing microorganism does not include a photosyntheticmicroorganism, such as algae. In certain embodiments, the C₁metabolizing microorganism will be an “obligate C₁ metabolizingmicroorganism,” meaning its sole source of energy comprises C₁substrates and nothing else.

As used herein, the term “methylotrophic bacteria” refers to anybacteria capable of oxidizing organic compounds that do not containcarbon-carbon bonds. In certain embodiments, a methylotrophic bacteriummay be a methanotroph. For example, “methanotrophic bacteria” refers toany methylotrophic bacteria having the ability to oxidize methane as itsprimary source of carbon and energy. Exemplary methanotrophic bacteriainclude Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylocystis, Methylomicrobium, or Methanomonas. In certain otherembodiments, the methylotrophic bacterium is an “obligate methylotrophicbacterium,” which refers to bacteria that are limited to the use of C₁substrates for the generation of energy.

In one specific embodiment of the invention, the process is performedusing methanotrophic bacteria of the type described in WO 02/18617 toproduce carotenoids, e.g., antheraxanthin, adonixanthin, astaxanthin,canthaxanthin, zeaxanthin and the other carotenoids mentioned on pages39 and 40 of WO 02/18617. To this end, the methanotrophic bacteriumMethylomonas 16a (ATCC PTA 2402) may particularly suitably be used.Carotenoids produced in this way may be separated out from the liquidculture medium as described in WO 02/18617, WO 02/20728 and WO 02/20733.

As used herein, the term “syngas” refers to a mixture including at leastcarbon monoxide (CO) and hydrogen (H₂). In at least some instances,syngas may also include CO₂, methane, and other gases in smallerquantities relative to CO and H₂. Syngas may be prepared using anyavailable process, including but not limited to, a water gas shift orcoal gasification process.

As used herein, the term “growth” is defined as any increase in cellmass. This may occur through cell division (replication) and theformation of new cells during “balanced growth,” or during “unbalancedgrowth” when cellular mass increases due to the accumulation of one ormore intracellular or intercellular polymers, such as certain lipids. Inthe latter case, growth may be manifest as an increase in cell size dueto the accumulation of a biopolymer within the cell. During “balancedcell growth,” all of the feedstocks (electron donors and electronacceptors) and all of the nutrients are present in the ratios requiredto make all of the macromolecular components of a cell. That is, nofeedstock or nutrient limits the synthesis of proteins, complexcarbohydrate polymers, fats, or nucleic acids. In contrast, during“unbalanced cell growth,” a feedstock or nutrient needed to make one ormore of a cell's macromolecules is not present in an amount or ratiorequired for balanced growth. Accordingly, this feedstock or nutrientbecomes limiting and is referred to as a “limiting nutrient.” Some cellsmay still achieve net growth under unbalanced conditions, but the growthis unbalanced and polymers that can be synthesized in the absence of thelimiting feedstock or nutrient will accumulate. These polymers includelipids or intracellular storage products, such as thepolyhydroxyalkanoates (PHAs), including polyhydroxybutyrate (PHB),polyhydroxyvalerate (PHV), and polyhydroxyhexanoate (PHHx)-glycogen, orsecreted materials, such as extracellular polysaccharide. Such oilcompositions are useful in the production of bioplastics.

Sample balanced and unbalanced growth conditions may differ in thenitrogen content in the media. For example, nitrogen constitutes about12% of dry cell weight, which means that 12 mg/L nitrogen must besupplied (along with a feedstock and other nutrients in the requiredstoichiometric ratios) to grow 100 mg/L dry cell weight. If otherfeedstock and nutrients are available in the quantities needed toproduce 100 mg/L of dry cell weight, but less than 12 mg/L nitrogen isprovided, then unbalanced cell growth may occur, with accumulation ofpolymers that do not contain nitrogen. If nitrogen is subsequentlyprovided, the stored polymer may serve as feedstock for the cell,allowing balanced growth, with replication and production of new cells.

As used herein, the term “growth cycle” as applied to a cell ormicroorganism refers to the metabolic cycle through which a cell ormicroorganism moves in culture conditions. For example, the cycle mayinclude various stages, such as a lag phase, an exponential phase, theend of exponential phase, and a stationary phase.

As used herein, the term “exponential growth,” “exponential phasegrowth,” “log phase” or “log phase growth” refer to the rate at whichmicroorganisms are growing and dividing. For example, during log phase,microorganisms are growing at their maximal rate given their geneticpotential, the nature of the medium, and the conditions under which theyare grown.

Microorganism rate of growth is constant during exponential phase andthe microorganism divides and doubles in number at regular intervals.Cells that are “actively growing” are those that are growing in logphase. In contrast, “stationary phase” refers to the point in the growthcycle during which cell growth of a culture slows or even ceases.

As used herein, the term “high growth variant” refers to an organism,microorganism, bacterium, yeast, or cell capable of growth with a C₁substrate, such as methane or methanol, as the sole carbon and energysource and which possesses an exponential phase growth rate that isfaster than the parent, reference or wild-type organism, microorganism,bacterium, yeast, or cell—that is, the high growth variant has a fasterdoubling time and consequently a high rate of growth and yield of cellmass per gram of C₁ substrate metabolized as compared to a parent cell(see, e.g., U.S. Pat. No. 6,689,601).

As used herein, the term “biofuel” refers to a fuel at least partiallyderived from “biomass.”

As used herein, the term “biomass” or “biological material” refers toorganic material having a biological origin, which may include one ormore of whole cells, lysed cells, extracellular material, or the like.For example, the material harvested from a cultured microorganism (e.g.,bacterial or yeast culture) is considered the biomass, which can includecells, cell membranes, cell cytoplasm, inclusion bodies, productssecreted or excreted into the culture medium, or any combinationthereof. In certain embodiments, biomass comprises the C₁ metabolizingmicroorganisms of this disclosure together with the media of the culturein which the C₁ metabolizing microorganisms of this disclosure weregrown. In other embodiments, biomass comprises C₁ metabolizingmicroorganisms (whole or lysed or both) of this disclosure recoveredfrom a culture grown on a C₁ (e.g., natural gas, methane). In stillother embodiments, biomass comprises the spent media supernatant orgases excreted or secreted from a culture of C₁ metabolizingmicroorganism culture on a C₁ substrate. Such a culture may beconsidered a renewable resource.

As used herein, the term “biorefinery” refers to a facility thatintegrates biomass conversion processes and equipment to produce fuelsfrom biomass.

As used herein, “oil composition” refers to the lipid content of abiomass (e.g., bacterial culture), including fatty acids, fatty acidesters, triglycerides, phospholipids, poly hydroxyalkanoates, isoprenes,terpenes, or the like. In oil composition of a biomass may be extractedfrom the rest of the biomass materials, such as by hexane or chloroformextraction. In addition, an “oil composition” may be found in any one ormore areas of a culture, including the cell membrane, cell cytoplasm,inclusion bodies, secreted or excreted into the culture medium, or anycombination thereof. An oil composition is neither natural gas nor crudepetroleum.

As used herein, the term “refinery” refers to an oil refinery, oraspects thereof, at which oil compositions (e.g., biomass, biofuel, orfossil fuels such as crude oil, coal or natural gas) may be processed.Sample processes carried out at such refineries include cracking,transesterification, reforming, distilling, hydroprocessing,isomerization, or any combination thereof.

As used herein, the terms “recombinant” or “non-natural” refer to anorganism microorganism, cell, nucleic acid molecule, or vector that hasat least one genetic alteration or has been modified by the introductionof a heterologous nucleic acid molecule, or refers to a cell that hasbeen altered such that the expression of an endogenous nucleic acidmolecule or gene can be controlled. Recombinant also refers to a cellthat is derived from a cell having one or more such modifications. Forexample, recombinant cells may express genes or other nucleic acidmolecules that are not found in identical form within the native cell(i.e., unmodified or wild type cell), or may provide an alteredexpression pattern of endogenous genes, such genes that may otherwise beover-expressed, under-expressed, minimally expressed, or not expressedat all. In another example, genetic modifications to nucleic acidmolecules encoding enzymes or functional fragments thereof can providebiochemical reaction(s) or metabolic pathway capabilities to arecombinant microorganism or cell that is new or altered from itsnaturally occurring state.

As used herein, the term “heterologous” nucleic acid molecule, constructor sequence refers to a nucleic acid molecule or portion of a nucleicacid molecule sequence that is not native to a cell in which it isexpressed or is a nucleic acid molecule with an altered expression ascompared to the native expression levels in similar conditions. Forexample, a heterologous control sequence (e.g., promoter, enhancer) maybe used to regulate expression of a gene or a nucleic acid molecule in away that is different than the gene or a nucleic acid molecule isnormally expressed in nature or culture. Generally, heterologous nucleicacid molecules are not endogenous to the cell or part of the genome inwhich they are present, and have been added to the cell by conjugation,transformation, transfection, electroporation, or the like.

As used herein, the term “vertical” refers to a direction that isaligned with the gravity vector at the location in question.

As used herein, the term “horizontal” refers to a direction that isperpendicular to the gravity vector at the location in question.

As used herein, the phrase “substantially vertical” refers to directionthat is less than 20° from vertical.

As used herein, the phrase “substantially horizontal” refers to adirection that is less than 20° from horizontal.

As used herein, the term “dished” refers to elliptical dished heads orends of the type used with pressure vessels, dished heads that meet ASMEstandards such as ASME 80:10 and standard flanged heads. A dished heador end is not conical.

The systems for fermentation of the instant disclosure may includeseparate units (e.g., processing units or systems that are disposed inclose proximity or adjacent to each other, or not), integrated units, orthe system itself may be interconnected and integrated. The systems ofthis disclosure may use at least one gas phase feedstock, including oneor more C₁ compounds, oxygen, and/or hydrogen. In certain embodiments,the fermentation system uses a C₁ metabolizing microorganism (e.g., amethanotroph such as Methylosinus trichosporium OB3b, Methylococcuscapsulatus Bath, Methylomonas sp. 16a, Methylomicrobium alcaliphilum20Z, or high growth variants or combinations thereof) as the primarymicroorganism in the fermentation culture.

A variety of culture methodologies may be used for the microorganism,bacteria and yeast described herein. For example, C₁ metabolizingmicroorganisms, such as methanotroph or methylotroph bacteria, may begrown by batch culture and continuous culture methodologies. Generallycells in log phase are often responsible for the bulk production of aproduct or intermediate of interest in some systems, whereas stationaryor post-exponential phase production can be obtained in other systems.

A classical batch culturing method is a closed system in which the mediacomposition is set when the culture is started and is not altered duringthe culture process. That is, media is inoculated at the beginning ofthe culturing process with one or more microorganisms of choice and thenis allowed to grow without adding additional microorganisms to thesystem. As used herein, a “batch” culture is in reference to notchanging the amount of a particular carbon source initially added,whereas control of factors such as pH and oxygen and/or hydrogenconcentration can be monitored and altered during the culture. In batchsystems, metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. Within batchcultures, cells (e.g., bacteria such as methylotrophs) will generallymove from a static lag phase to a high growth logarithmic phase to astationary phase where growth rate is reduced or stopped (and willeventually lead to cell death if conditions do not change).

A fed-batch system is a variation on the standard batch system in whicha carbon substrate of interest is added in increments as the cultureprogresses. Fed-batch systems are useful when cell metabolism is likelyto be inhibited by catabolite repression and when it is desirable tohave limited amounts of substrate in the media. Since it is difficult tomeasure actual substrate concentration in fed-batch systems, an estimateis made based on changes of measurable factors such as pH, dissolvedoxygen, and the partial pressure of waste gases. Batch and fed-batchculturing methods are common and known in the art (see, e.g., Thomas D.Brock, Biotechnology: A Textbook of Industrial Microbiology, 2nd Ed.(1989) Sinauer Associates, Inc., Sunderland, Mass.; Deshpande, 1992,Appl. Biochem. Biotechnol. 36:227).

Continuous cultures are “open” systems in the sense that defined culturemedia is continuously added to a bioreactor while an equal amount ofused (“conditioned”) media is removed simultaneously for processing.Continuous cultures generally maintain the cells at a constant high,liquid phase density where cells are primarily in logarithmic growthphase. Alternatively, continuous culture may be practiced withimmobilized cells (e.g., biofilm) where carbon and nutrients arecontinuously added and valuable products, by-products, and wasteproducts are continuously removed from the cell mass. Cellimmobilization may be achieved with a wide range of solid supportscomposed of natural materials, synthetic materials, or a combinationthereof.

Continuous or semi-continuous culture allows for the modulation of oneor more factors that affect cell growth or end product concentration.For example, one method may maintain a limited nutrient at a fixed rate(e.g., carbon source, nitrogen) and allow all other parameters to changeover time. In other embodiments, several factors affecting growth may becontinuously altered while cell concentration, as measured by mediaturbidity, is kept constant. The goal of a continuous culture system isto maintain steady state growth conditions while balancing cell loss dueto media being drawn off against the cell growth rate. Methods ofmodulating nutrients and growth factors for continuous culture processesand techniques for maximizing the rate of product formation are wellknown in the art (see Brock, 1992).

In certain embodiments, culture media includes a carbon substrate as asource of energy for a C₁ metabolizing microorganism. Suitablesubstrates include C₁ substrates, such as methane, methanol,formaldehyde, formic acid (formate), carbon monoxide, carbon dioxide,methylated amines (methylamine, dimethylamine, trimethylamine, etc.),methylated thiols, or methyl halogens (bromomethane, chloromethane,iodomethane, dichloromethane, etc.). In certain embodiments, culturemedia may comprise a single C₁ substrate as the sole carbon source for aC₁ metabolizing microorganism, or may comprise a mixture of two or moreC₁ substrates (mixed C₁ substrate composition) as multiple carbonsources for a C₁ metabolizing microorganism.

Additionally, some C₁ metabolizing organisms are known to utilize non-C₁substrates, such as sugar, glucosamine or a variety of amino acids formetabolic activity. For example, some Candida species can metabolizealanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489,1990). Methylobacterium extorquens AM1 is capable of growth on a limitednumber of C₂, C₃, and C₄ substrates (Van Dien et al., Microbiol.149:601-609, 2003). Alternatively, a C₁ metabolizing microorganism maybe a recombinant variant having the ability to utilize alternativecarbon substrates. Hence, it is contemplated that a carbon source inculture media may comprise a mixture of carbon substrates, with singleand multi-carbon compounds, depending on the C₁ metabolizingmicroorganism selected.

In certain embodiments, the instant disclosure provides a method formaking fuel, comprising converting biomass from a culture primarilycomprising a C₁ metabolizing non-photosynthetic microorganism into anoil composition and refining the oil composition into a fuel. In certainembodiments, the C₁ metabolizing non-photosynthetic microorganism is anobligate C₁ metabolizing non-photosynthetic microorganism, such as anobligate methanotroph or methylotroph. In further embodiments, the C₁metabolizing non-photosynthetic microorganism is a recombinantmicroorganism comprising a heterologous polynucleotide encoding a fattyacid producing enzyme, a formaldehyde assimilation enzyme, or acombination thereof. In further embodiments, the oil composition isderived or extracted from cell membrane of the C₁ metabolizingnon-photosynthetic microorganism, such as a methylotroph ormethanotroph.

In certain embodiments, the instant disclosure provides a method formaking fuel by refining an oil composition in a refining unit to producefuel, wherein the oil composition is derived from a C₁ metabolizingnon-photosynthetic microorganism, such as a methylotroph ormethanotroph. In further embodiments, the method further comprises useof a processing unit for extracting the oil composition from the C₁metabolizing non-photosynthetic microorganism. In still furtherembodiments, the method comprises (a) culturing C₁ metabolizing bacteriain the presence of a feedstock comprising a C₁ substrate in a controlledculturing unit, wherein the cultured bacteria produces an oilcomposition; (b) extracting the oil composition from the culturedbacteria in a processing unit; and (c) refining the extracted oilcomposition in a refining unit to produce fuel. In certain embodiments,the feedstock C₁ substrate is methane, methanol, formaldehyde, formicacid, carbon monoxide, carbon dioxide, a methylamine, a methylthiol, ora methylhalogen.

In certain embodiments, the instant disclosure provides a method formaking natural products, such as ethanol, acetate, butanol, single-cellprotein, sugars, or other metabolites or cellular products wherein thenatural product is derived from a C₁ metabolizing non-photosyntheticmicroorganism, such as a methylotroph or methanotroph.

In further embodiments, the method further comprises use of a processingunit for extracting the natural product from the C₁ metabolizingnon-photosynthetic microorganism.

In still further embodiments, the method comprises (a) culturing C₁metabolizing bacteria in the presence of a feedstock comprising a C₁substrate in a controlled culturing unit, wherein the cultured bacteriaproduce a natural product; (b) extracting the natural product from thecultured bacteria in a processing unit; and (c) refining the naturalproduct to produce a commercial product. In certain embodiments, thefeedstock C₁ substrate is methane, methanol, formaldehyde, formic acid,carbon monoxide, carbon dioxide, a methylamine, a methylthiol, or amethylhalogen.

In certain embodiments, the instant disclosure provides a method formaking natural or non-natural products, such as ethanol, acetate,butanol, isoprene, propylene, farnesene, enzymes, or other metabolitesor cellular products wherein the product is derived from a geneticallyengineered C₁ metabolizing non-photosynthetic microorganism, such as amethylotroph or methanotroph which has been transformed with aheterologous nucleotide sequence. In further embodiments, the methodfurther comprises use of a processing unit for extracting the productfrom the genetically engineered C₁ metabolizing non-photosyntheticmicroorganism. In still further embodiments, the method comprises (a)culturing genetically engineered C₁ metabolizing bacteria in thepresence of a feedstock comprising a C₁ substrate in a controlledculturing unit, wherein the cultured bacteria produce a natural product;(b) extracting the natural product from the cultured bacteria in aprocessing unit; and (c) refining the natural product to produce acommercial product. In certain embodiments, the feedstock C₁ substrateis methane, methanol, formaldehyde, formic acid, carbon monoxide, carbondioxide, a methylamine, a methylthiol, or a methylhalogen.

In certain embodiments, the instant disclosure provides a method formaking natural or non-natural products, such as ethanol, acetate,butanol, isoprene, propylene, farnesene, enzymes, or other metabolitesor cellular products wherein the product is derived from a non-C₁metabolizing microorganism, such as Escherichia coli, Saccaromycescerevisiae, or other common production microorganism. In certainembodiments, the feedstock substrate is glucose, sucrose, glycerol,cellulose or other multicarbon feedstocks.

An exemplary system for stimulating production of biomass in accordancewith embodiments described herein includes a loop reactor 100 of thetype illustrated in FIG. 1. Loop reactor 100 illustrated in FIG. 1includes a substantially vertical downflow zone 103 and a substantiallyvertical upflow zone 105 separated by a substantially horizontal zone orloop section identified by the plurality of brackets 104. The outlet ofsubstantially vertical downflow zone 103 is in fluid communication withan inlet of fluid moving device 107. The outlet of fluid moving device107 is in fluid communication with the inlet of substantially horizontalzone 104. The outlet of substantially horizontal zone 104 is in fluidcommunication with the inlet of substantially vertical upflow zone 105.The outlet of substantially vertical upflow zone 105 is in fluidcommunication with an inlet of gas/liquid separation vessel 109. Anoutlet of gas/liquid separation vessel 109 is in fluid communicationwith the inlet of substantially vertical downflow zone 103 via a drainconduit having an inlet end connected to the gas/liquid separationvessel 109 and having an outlet end connected to the substantiallyvertical downflow zone 103. Specific embodiments of systems forstimulating production of biomass in accordance with embodimentsdescribed herein includes a fluid conduit of non-increasing diameterextending between the outlet of the gas/liquid separation vessel 109 andthe inlet of fluid moving device 107. Fluid flows through loop reactor100 in a clockwise direction (as indicated by the arrows) under theinfluence of fluid moving device 107.

FIG. 2 shows an exemplary system 200 for stimulating production ofbiomass that includes a loop reactor 100 along with an optionalseparation subsystem 250, an optional thermal subsystem 270 and optionalcontrol subsystem 290. Although shown as an integrated system 200, theoptional subsystems may be installed or otherwise combined with the loopreactor 100 either individually or in any combination. One or moreliquids and one or more gas substrates are introduced to the loopreactor 100 to form a multi-phase mixture with a liquid culture mediathat travels through the loop reactor 100. After passage through theloop reactor 100, the multi-phase mixture may contain one or morecompounds produced by the biological organisms within the loop reactor100, unconsumed nutrients and other compounds in the liquid within themulti-phase mixture, unconsumed gases in the gas bubbles within themulti-phase mixture, and microbiological organisms in the form ofbiosolids. Excess microbiological organisms may be removed from the loopreactor 100 as biomass either intermittently or continuously. Biomassaccumulations within the loop reactor 100 may be removed to maintain theoverall biomass within the loop reactor 100 within a defined range orabove or below a defined threshold. In at least some instances, biomassremoved from the loop reactor 100 may include one or more usefulcompounds. For example, the biological organisms within the excessbiomass may contain an amount of one or more intracellular lipids orsimilar compounds useful in the production of a biofuel such asbiodiesel or protein-containing products.

The one or more liquids may include any liquid suitable for sustainingor delivering one or more nutrients to the microbiological organismswithin the loop reactor 100. Such liquids may include, but are notlimited to, solutions containing water, one or more alcohols, minerals,one or more nitrogen-containing compounds, one or morephosphorus-containing compounds, and the like. In at least someinstances, one or more fluid movers are used to deliver the one or moreliquids to the loop reactor 100 in a controlled manner and pressure. Theone or more fluid movers can include any type of pump or similar devicecapable of transferring a liquid between two points. Example fluidmovers include, but are not limited to, centrifugal pumps, positivedisplacement pumps, progressing cavity pumps, double diaphragm pumps,and the like. Other illustrative fluid movers include, but are notlimited to eductors, ejectors, and similar devices. The transfer ofliquid to the loop reactor 100 can be flow controlled, pressurecontrolled, or controlled using combinations of pressure, temperature,flow, level, flowrate, superficial velocity, or compositional analysisprocess variable data gathered from one or more points within the loopreactor 100 or from one or more points within the system 200. In atleast some instances, the transfer of liquid by the fluid mover can becontrolled based on the measured concentration of one or more componentsor compounds (e.g., one or more carbon-containing or nitrogen-containingnutrients) within the loop reactor 100; for example, the flow of liquidtransferred by the fluid mover may be increased in response to ameasured decrease in nutrient concentration within the loop reactor 100.

The one or more gas substrates can include any gas, gases, orcombination of gases suitable for sustaining or delivering one or morenutrients to the biological organisms within the loop reactor 100. Suchgases can include, but are not limited to, one or more gases containingcarbon compounds. Such gases can include, but are not limited to, one ormore gases containing C₁ carbon compounds such as methane or carbonmonoxide. The one or more gas substrates may also include one or moregases used in the metabolic processes of the biological organisms withinthe loop reactor 100. Such gases can include, but are not limited to,oxygen, oxygen-containing compounds and hydrogen. The one or more gassubstrates may be transferred to the loop reactor 100 as a pure gas oras a gas mixture (e.g., syngas, a mixture of carbon monoxide andhydrogen). The one or more gas substrates may be transferred to the loopreactor 100 individually (e.g., methane and an oxygen-containing gassuch as air may be transferred individually to minimize the likelihoodof formation of an explosive gas mixture external to the loop reactor100).

The one or more gas substrates may optionally be transferred to the loopreactor 100 using a gas mover. Example gas movers include, but are notlimited to, rotary lobe compressors, centrifugal compressors, screwcompressors, and the like. The delivery pressure of the one or more gassubstrates depends upon a variety of factors including the operatingpressure of the loop reactor 100 and the pressure drop associated withthe gas distributor used to distribute the one or more gas substrateswithin the loop reactor 100. Similarly, the delivery flowrate of the oneor more gas substrates may be manually or automatically controlled tomaintain the concentration or level of dissolved gas within the loopreactor 100 within a defined range (e.g., dissolved oxygen above atleast 4 ppm) based at least in part on the needs of the biologicalorganisms present in the loop reactor 100. In at least some instances,the one or more gas substrates can be delivered to the loop reactor 100at a pressure of from about 5 psig to about 600 psig; from about 25 psigto about 400 psig; or from about 50 psig to about 300 psig.

Any number of gases may be introduced through a common gas distributionheader or any number of individual gas distribution headers. Such gasdistribution headers may introduce all of the gas substrate at a singlepoint within the loop reactor 100 or may introduce portions of the gassubstrate at various locations throughout the loop reactor 100. In atleast some instances, the gas substrate can include, but is not limitedto, methane, carbon monoxide, hydrogen, or oxygen. In at least someinstances, the feed rate of the gas substrate can be referenced to thefeed rate of the liquid media. For example, methane may be introduced asa gas substrate at a rate of from about 0.1 grams of methane/liter ofliquid media (g/l) to about 100 g/l; from about 0.5 g/l to about 50 g/l;or from about 1 g/l to about 25 g/l. Carbon monoxide (“CO”) may beintroduced as a gas substrate 204 at a rate of from about 0.1 grams ofCO/liter of liquid media (g/l) to about 100 g/l; from about 0.5 g/l toabout 50 g/l; or from about 1 g/l to about 25 g/l. Oxygen may beintroduced as a gas substrate 204 at a rate of from about 1 grams ofoxygen/liter of liquid media (g/l) to about 100 g/l; from about 2 g/l toabout 50 g/l; or from about 5 g/l to about 25 g/l. Hydrogen may beintroduced as a gas substrate 204 at a rate of from about 0.01 grams ofhydrogen/liter of liquid media (g/l) to about 50 g/l; from about 0.1 g/lto about 25 g/l; or from about 1 g/l to about 10 g/l.

Within the loop reactor 100 the microbiological organisms willmetabolize at least a portion of the carbon-containing compounds presentin the multi-phase mixture. At least a portion of this process mayinclude the production of additional microbiological organisms thatincrease the overall quantity of biomass present in the loop reactor100. Left uncontrolled, the biomass within the loop reactor 100 mayaccumulate to a point such that one or more operational aspects of theloop reactor 100 (e.g., flowrate, pressure drop, production of desiredproducts, etc.) is compromised or adversely affected by the presence ofthe excess biomass. In such instances, the ability to remove at least aportion of the biomass present in the loop reactor 100 is desirable. Inat least some instances, biomass preferentially accumulates at alocation within a gas/liquid separation vessel (109 in FIGS. 3-9)facilitating biosolids removal from the loop reactor 100 via at leastone biomass removal port provided in gas/liquid separation vessel 109 orat a different location or locations along loop reactor 100. The removedbiomass can be delivered to separation subsystem 250 where the biomasscan be further processed and desirable products recovered from thebiomass.

In at least some instances, all or a portion of the biomass productionprocess may be at least partially automatically controlled using acontrol subsystem 290. The control subsystem 290 may collectprocess-related information provided by one or more process elements inthe form of signals containing analog or digital data representing oneor more process variables. For instance, the control subsystem cancollect process-related signals using one or more process elementsincluding, but not limited to, mass flow sensors, volumetric flowsensors, temperature sensors, pressure sensors, level sensors,analytical sensors (e.g., dissolved oxygen sensors, methane sensors,ammonia sensors, biological oxygen demand or “BOD” sensors, pH sensors,conductivity sensors, and the like) or any other device capable ofproviding a signal containing data representative of one or moreprocess-related conditions within the loop reactor 100.

The control subsystem 290 may execute one or more sets of instructionscontrolling, altering, or adjusting one or more aspects of thefermentation process based at least in part on the process variablesignals received from the process elements. Such instructions may resultin the generation of one or more control output signals by the controlsubsystem 290. The control output signals can be transmitted from thecontrol subsystem 290 to one or more final control elements such asblock valves, control valves, motors, variable speed drives, thermalenergy sources or sinks, etc. The interaction between the final controlelements and the fermentation process can, in turn, provide the controlsubsystem 290 with a high degree of relatively accurate control of thebiomass production process.

For example, responsive to the receipt of one or more signals containingdata indicative of the temperature of the multi-phase mixture in theloop reactor 100, the control subsystem 290 may initiate, alter, orcease the flow of thermal transfer media to a heat transfer unitoperation. Similarly, responsive to the receipt of one or more signalscontaining data indicative of the dissolved oxygen level of themulti-phase mixture in the loop reactor 100, the control subsystem 290may increase, decrease, or maintain the flow of the oxygen-containinggas substrate to the loop reactor 100. Although only two illustrativeexamples are provided herein, any flow, level, pressure, analyticalvalue, or the like that is appropriate to the fermentation process maybe similarly controlled by the control subsystem 290 using one or moreappropriate process sensors and one or more appropriate final controlelements.

Exemplary system 200 in additional embodiments includes othersubsystems, including a nutrient and/or a mineral supply subsystem and aheat transfer unit operation(s). Exemplary system 200 stimulatesproduction of biomass by introducing gaseous substrate(s) andnutrient(s) to a liquid culture medium to form a multi-phase mixture ofthe liquid culture medium, supplied gaseous substrate(s) and nutrient(s)within loop reactor 100. This multi-phase mixture flows through loopreactor 100 by the action of fluid flow unit operation 107. The liquidculture medium includes microorganisms capable of converting gaseoussubstrates to desirable products, some of which may be recovered fromthe microorganisms or from the gas phase and/or liquid phase that formin gas/liquid separation unit operation 109. Gaseous substrate(s) andnutrient(s) can be delivered to loop reactor 100 from nutrient supplysubsystem, and loop reactor 100 is operated under conditions thatpromote mass transfer of gaseous substrate(s) and nutrient(s) into theliquid culture medium and into the microorganisms. Nutrients andminerals can be introduced into loop reactor 100 at one or morelocations. Gas/liquid separation vessel 109 receives the liquid culturemedium, including any gases that remain in the liquid culture medium,and gases which have separated from the liquid culture medium. Withingas/liquid separation vessel, the multi-phase liquid mixture separatesinto at least a liquid phase and a gas phase.

FIGS. 3-9 show an exemplary gas/liquid separation vessel 109 useful in aloop reactor 100 of system 200 for stimulating production of biomass.Exemplary system 200 includes a loop reactor 100 including a gas/liquidseparation unit operation 109 (e.g., a gas/liquid separation vessel)where gases separate from liquid of the multi-phase mixture of liquidculture media including microorganisms and gases flowing through loopreactor 100. Elements of loop reactor 100 including but not limited togas/liquid separation unit operation 109, e.g., a gas/liquid separationvessel 109, fluid flow unit operation 107 (e.g., pump or other devicecapable of causing a fluid to move), substantially horizontal zone 104,substantially vertical downflow zone 103 and substantially verticalupflow zone 105 can be a metallic, non-metallic, or composite structure.For example, the elements can include one or more metallic materialssuch as 304, 304L, 316, or 316L stainless steels. In some instances, oneor more coatings, layers, overlays, inserts, or other materials can bedeposited on, applied to, joined with, or formed integral to all or aportion of the metallic, non-metallic or composite structures tobeneficially or detrimentally affect the ability for microbiologicalorganisms to attach thereto or to grow thereupon. For example, a coatinginhibiting the growth or attachment of microbiological organisms may bedeposited on or formed integral with the surfaces of the loop reactor100 that are thermally conductively coupled to a heat transfer unitoperation. In another example, a coating that inhibits the growth orattachment of biological organisms may be deposited on or formedintegral with portions of loop reactor 100 where it is desired toachieve removal of accumulated biomass more easily.

In at least some instances, the construction of elements of loop reactor100 can include features that facilitate sterilization of all or aportion of the process contact surfaces. Such sterilization can beaccomplished for example using steam sterilization, ultravioletsterilization, chemical sterilization, or combinations thereof. In atleast some instances, one or more non-metallic materials or one or morenon-metallic coatings may be used within all or a portion of theinterior or exterior of some or all of the elements of loop reactor 100.The use of such non-metallic materials may advantageously provide, forexample, sterilizable surfaces that are capable of supporting orpromoting biological growth.

Within gas/liquid separation vessel 109 the multi-phase mixtureseparates into at least a gas effluent and a liquid effluent. In atleast some instances, biosolids present in the multi-phase mixture maybe separated into a solids-containing effluent within gas/liquidseparation vessel 109. In at least some instances, at least a portion ofthe solids-containing effluent from the gas/liquid separation vessel 109can be combined with the one or more liquids and the mixture returned togas/liquid separation vessel 109, loop section 104, substantiallyvertical downflow zone 103 or substantially vertical upflow zone 105. Inat least some instances, system 200 can include one or more gas/liquidseparators 109 operating in parallel or series.

Referring to FIGS. 3 and 4, gas/liquid separation vessel 109 inaccordance with embodiments described herein may be a longitudinal,horizontal vessel through which the multi-phase mixture flows in ahorizontal direction. Gas/liquid separation vessel 109 includes anintermediate section 301, an outlet end section 303 and an inlet endsection 305. Outlet end section 303 is located on the downstream side ofintermediate section 301 while inlet end section 305 is located on theopposite side of intermediate section 301 upstream of intermediatesection 301. In FIGS. 3 and 4, the multi-phase mixture flows throughgas/liquid separation vessel 109 in the direction of arrow 307 in FIG.3. Outlet end section 303 includes outlet 309 from gas/liquid separationvessel 109 and inlet end section 305 includes inlet 311 into gas/liquidseparation vessel 109. Further details of intermediate section 301,outlet end section 303 and inlet end section 305 are described below.

In the illustrated embodiments shown in FIGS. 3-9, intermediate section301 may be a non-rectangular, cylindrical member having a constantdiameter D and a horizontal centerline defining a longitudinal axis 302of gas/liquid separation vessel 109. An outlet 312 of intermediatesection 301 is located at a downstream end of intermediate section 301and an inlet 314 to intermediate section 301 is located at an upstreamend of intermediate section 301. Diameter D can vary, with exemplarydiameters D ranging from about 2 meters to about 8 meters and 3 metersto 6 meters; however, in other embodiments, diameter D may be less than2 meters or more than 8 meters or less than 3 meters or more than 6meters. Inlet 314 of intermediate section 301 and outlet 312 ofintermediate section 301 are spaced apart by a length L_(i). L_(i) canvary, with exemplary values for L_(i) ranging from about 2 meters toabout 8 meters and 3 meters to 7 meters; however, in other embodiments,L_(i) may be less than 2 meters or more than 8 meters and less than 3meters or more than 7 meters. In the illustrated embodiment shown inFIGS. 3-9, intermediate section 301 is illustrated as being round inshape; however, intermediate section 301 is not limited to a roundshape. For example, intermediate section 301 can have othernon-rectangular shapes in a vertical cross-section.

Continuing to refer to FIGS. 3-9, outlet side section 303 includes adished end 313 at the downstream end of gas/liquid separation vessel109. Outlet side section 303 opposite dished end 313 includes an inletend 315, which coincides with and is in fluid communication with outlet312 of intermediate section 301. In the embodiment illustrated in FIGS.3-9, inlet 315 of outlet side section 303 has a diameter that is equalto diameter D of intermediate section 301. While an embodiment ofgas/liquid separation vessel 109 has been illustrated in FIGS. 3-9 witha dished end, gas/liquid separation vessel 109 is not limited to anoutlet side section 303 that includes a dished end 313. In accordancewith other embodiments of gas/liquid separation vessel 109, outlet sidesection 303 does not include a dished end 313. For example, the end ofoutlet side section 303 may have a shape that is not dished, forexample, the end of the outlet side section 303 may not be dished andmay be non-conical, e.g., flat or planar or other shape. Outlet sidesection 303 in the embodiment illustrated in FIGS. 3-9 has a lengthL_(o) that is less than length L_(i). L_(o) can vary, for example, L_(o)may range from about 0.5 meters to about 3 meters; however, in otherembodiments, L_(o) may be less than 0.5 meters or greater than about 3meters. Dished end 313 includes an outlet or drain 309 in fluidcommunication with an inlet of substantially vertical downflow zone 103.Outlet 309 is located in a lower half or lower portion of dished end313. Outlet 309 is connected to an inlet of substantially vertical downflow zone 103 by a 90° bend or drain conduit 317. Bend 317 has a radiuswhich can vary, with exemplary radiuses ranging from about 0.5 to 3times the diameter of the substantially vertical downflow zone 103;however, the radius of bend 317 can be less than 0.5 or more than 3times the diameter of substantially vertical downflow zone 103. Inaccordance with embodiments described herein, outlet 309 of dished end313 is in fluid communication with fluid moving device 107 via bend 317and substantially vertical downflow zone 103 and the diameter of bend317 and substantially vertical downflow zone 103 is non-increasing,i.e., diameter of bend 317 and substantially vertical downflow zone 103is a constant or does not increase in diameter. In the illustratedembodiment, an inlet of bend or drain conduit 317 is in fluidcommunication with the outlet or drain 309 of dished end 313 and anoutlet of bend or drain conduit 317 is in fluid communication with theinlet of vertical downflow zone 103.

Inlet side section 305 has the shape of an oblique conical frustum. Asused herein the phrase “conical frustum” refers to a frustum (i.e.,portion of a cone that lies between two parallel planes, e.g.,horizontal planes, cutting through the cone) wherein the truncated endsof the cone resulting from the two parallel planes cutting through thecone are non-rectangular, i.e., not rectangular. For example, inaccordance with embodiments described herein, both the truncated ends ofthe cone resulting from the two parallel planes cutting through the coneare circular or both are non-rectangular in shape. In other examples ofembodiments described herein, the truncated end of the cone which hasthe larger diameter resulting from one of the parallel planes cuttingthrough the cone is circular. As used herein, “cone” refers to thethree-dimensional geometric shape that tapers smoothly from a flat base(e.g., circular) to a point called the apex or vertex. “Oblique conicalfrustum” refers to a conical frustum where the centers of the truncatedends of the cone defined by the parallel planes that cut throughdifferent sections of the cone do not have their axis on the sameperpendicular, but instead two edges of the truncated ends of the conedefined by the parallel planes that cut through different portions ofthe cone are connected by the same perpendicular. In FIG. 4, inlet sidesection 305 includes an inlet 311 at an upstream end of inlet sidesection 305 and an outlet 319 at a downstream end of inlet side section305. Outlet 319 of inlet side section 305 coincides with and is of thesame diameter D as inlet 314 of intermediate section 301. In theembodiment shown in FIG. 4, inlet side section having the shape of anoblique conical frustum is lying on the edge that is common to each ofthe truncated ends of the cone that forms the oblique conical frustum.Outlet 319 defines one truncated end of the cone defined by one of theparallel planes that cuts through the cone and inlet 311 defines theother truncated end of the cone defined by the other parallel plane thatcuts through the cone so as to define a conical frustum. The center ofthe base of the cone (i.e., at outlet 319) and the center of the otherend of the cone (i.e., at inlet 311) making up the oblique conicalfrustum shape of inlet side section 305 do not lie in the sameperpendicular if inlet side section 305 were rotated 90° in acounterclockwise direction from the position illustrated in FIG. 4.Rotating inlet side section 305 90° in a counterclockwise direction fromthe position illustrated in FIG. 4 would result in inlet end section 305resting on the base of the cone (i.e., outlet 319) with the inlet 311spaced vertically from outlet 319. In such orientation, an edge of thebase of the cone (i.e., at outlet 319) defined by one parallel planethat cuts through the cone lies in the same perpendicular as an edge ofthe top of the cone (i.e., at the inlet 311) defined by the otherparallel plane that cuts through the cone so as to define an obliqueconical frustum. In the context of the orientation of inlet side section305 illustrated in FIG. 4, inlet 311 of inlet side section 305 includesa lowermost edge and outlet 319 of inlet end section 305 includes alower most edge that lie on, i.e., contact a common horizontal plane329. In the embodiment illustrated in FIG. 4, intermediate section 305includes a lowermost edge 325 and outlet end section 303 includes alowermost edge 327 that also lie on, i.e., contact common horizontalplane 329. The oblique conical frustum shape of inlet end section 305may also be characterized by cone angle α which ranges between about 5to 30°. While the cone angle α can range between 5 and 30°, in otherembodiments, the cone angle α may be less than 5° or greater than 30°.

Inlet 311 of inlet side section 305 has a diameter that is less than thediameter of outlet 319. Inlet 311 is in fluid communication with anoutlet of bend or feed conduit 321. An inlet of bend or feed conduit 321is in fluid communication with an outlet of substantially verticalupflow zone 105. Bend 321 has a radius which can vary, with exemplaryradiuses ranging from about 0.5 to 3 times the diameter of substantiallyvertical upflow zone 105; however, in other embodiments, the radius ofbend 321 can be less than 0.5 or more than 3 times the diameter ofsubstantially vertical upflow zone 105. Inlet 311 of inlet side section305 is spaced from outlet 319 of inlet side section 305 by a distanceL_(a). L_(a) can vary, for example, L_(a) may range from about 2 metersto about 10 meters or 4 meters to 8 meters; however, in otherembodiments, L_(a) may be less than 2 meters or greater than about 10meters or less than 4 meters or greater than 8 meters. The length L ofgas/liquid separation vessel 109 is equal to the sum of lengths L_(i),L_(o) and L_(a). A ratio of L_(o)/L can range from about 0.08 to 0.2;however, in other embodiments, the ratio of L_(o)/L can be less than0.08 or greater than 0.2. A ratio of L_(i)/L can range from about 0.3 to0.5; however, in other embodiments, the ratio of L_(i)/L can be lessthan 0.3 or greater than 0.5. A ratio of L_(a)/L can range from about0.4 to 0.6; however, in other embodiments, the ratio of L_(a)/L can beless than 0.04 or greater than 0.6.

When a multi-phase mixture is flowing through gas/liquid separationvessel 109 a gas head space exists above the multi-phase mixture withingas/liquid separation vessel 109. Gas which desorbs from the multi-phasemixture can collect in this gas head space. Specific embodiments ofgas/liquid separation vessels 109 in accordance with embodimentsdescribed herein are shaped and sized so that this gas head space ischaracterized by a ratio of hydraulic diameter (D_(h)) to length (L_(g))where D_(h) is the hydraulic diameter of the volume within gas/liquidseparation vessel 109 occupied by the gas head space and L_(g) is thelength of the interface between the multi-phase mixture and gas headspace within gas/liquid separation vessel 109. D_(h)=4A/P where A is thegas-wetted cross-sectional area of gas/liquid separation vessel 109 andP is the gas-wetted perimeter of the gas-wetted cross-section ofgas/liquid separation vessel 109. Ratios of D_(h) to L_(g) can varydepending on the size and shape of gas/liquid separation vessel 109 andthe level of the multi-phase mixture within the gas/liquid separationvessel 109. For example, when the depth of the multi-phase mixturewithin gas/liquid separation vessel 109 ranges between 50 to 90 percentof the diameter of intermediate section 301 of gas/liquid separationvessel 109, the ratio of D_(h) to L_(g) is selected to be less thanabout 5 or less than 5. Liquid effluent and biosolids removed fromgas/liquid separation vessel 109 may be received at inlet of fluidmoving device 107, e.g., a pump, and output from an outlet of fluidmoving device 107. Outlet of fluid moving device 107 is in fluidcommunication with inlet of loop section 104 of loop reactor 100.Suitable pumps for moving liquid effluent and biosolids include pumpscapable of moving fluids (liquids or gases) and slurries, by mechanicalaction and which are able to produce desired flow rates in thesubstantial absence of shear forces detrimental to the biomass and/orcavitation. Avoiding cavitation is desirable because cavitation causesgaseous substrates and nutrients in the multi-phase mixture to come outof solution making them less accessible to the biomass. Examples of suchtype of pumps are centrifugal pumps, although pumps which are notcentrifugal pumps may also be used. For example, positive displacementpumps, progressive cavity pumps, double diaphragm pumps, and the likecan also be used. Devices other than pumps can also be used to move themulti-phase mixture, for example, propellers driven by a motor, such asthe propellers and motors described in U.S. Pat. No. 7,579,163 can beused instead of or in combination with a pump.

In exemplary embodiments described herein, gas pressure in headspace ofgas/liquid separation unit operation 109 ranges from about 0.2 to about0.6 bars; however, gas pressure in the headspace is not limited to arange of about 0.2 to about 0.6 bars. For example, in exemplaryembodiments described herein, gas pressure in headspace can be less than0.2 bars or greater than about 0.6 bars. The pressure at the inlet offluid moving device 107 ranges from about 0.2 bars to about 4.0 bars;however, the pressure at the inlet of fluid moving device 107 is notlimited to a range of about 0.2 bars to about 4.0 bars. For example, inexemplary embodiments described herein, the pressure at the inlet offluid moving device 107 can be less than about 0.2 bars or greater thanabout 4.0 bars. The pressure at outlet of fluid moving device 107 rangesfrom about 3.5 bars to about 4.0 bars; however, the pressure at theoutlet of fluid moving device 107 is not limited to a range of about 3.5bars to about 4.0 bars. For example, in exemplary embodiments describedherein, the pressure at the outlet of fluid moving device 107 can beless than about 3.5 bars or greater than about 4.0 bars. In exemplaryembodiments that include static mixers in loop reactor 100, the pressuredrop across a static mixer ranges from about 0.03 to about 0.05 bars;however, the pressure drop across a static mixer is not limited to arange from about 0.03 to about 0.05 bars. For example, in exemplaryembodiments described herein, the pressure drop across a static mixermay be less than 0.03 bars or greater than 0.05 bars. In accordance withexemplary embodiments described herein, pressure within loop section 104at the bottom or beginning of substantially vertical upflow zone 105ranges from about 1.0 to about 3.0 bars; however, the pressure withinloop section 104 at the beginning of substantially vertical upflow zone105 is not limited to a range from about 1.0 to about 3.0 bars. Forexample, pressure within loop section 106 at the beginning ofsubstantially vertical upflow zone 105 may be less than about 1.0 barsor greater than about 3.0 bars. In accordance with exemplary embodimentsdescribed herein, pressure at the outlet/top of substantially verticalupflow zone 105 ranges from about 0.2 bars to about 0.6 bars; however,the pressure at outlet/top of substantially vertical upflow zone 105 isnot limited a range of about 0.2 bars to about 0.6 bars. For example, inaccordance with embodiments described herein, pressure at outlet/top ofsubstantially vertical upflow zone 105 can be less than about 0.2 barsor greater than about 0.6 bars. In embodiments described herein, thepressure drop across substantially vertical upflow zone 105 can rangefrom about 1.0 bars to about 2.3 bars; however, the pressure drop acrossthe substantially vertical upflow zone 105 is not limited to a rangefrom about 1.0 bars to about 2.3 bars. For example, the pressure dropacross the substantially vertical upflow zone 105 can be less than 1.0bars or more than 2.3 bars. In some instances, the pressure drop acrosssubstantially vertical upflow zone 105 accounts for at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70% or atleast 80% of the pressure drop between the outlet of fluid moving device107 and the headspace of gas/liquid separation vessel 109.

In at least some instances, gas effluent or the gas phase separated fromthe multi-phase mixture in gas/liquid separation vessel 109 may includea mixture of one or more gas substrates (e.g., methane or carbonmonoxide) and one or more gaseous byproducts (e.g., carbon dioxide)generated as a byproduct by the biological organisms in loop reactor100. In at least some instances, gas effluent may be separated from themulti-phased mixture in gas/liquid separation vessel 109 and at least aportion of the one or more gas substrates recycled (not shown) to theloop reactor 100, for example as a gas substrate. In at least someinstances, the gas effluent may include one or more useful compounds.For example, the gas effluent may contain an amount of one or moregaseous C₂₊ hydrocarbon compounds and compounds based thereupon havingvalue as either a finished product or as a raw material in a subsequentprocess. Such useful compounds may be separated from the gas effluentprior to recycling at least a portion of the gas effluent to loopreactor 100.

In at least some instances, liquid effluent or the liquid phaseseparated from the multi-phased mixture in gas/liquid separation vessel109 may include a mixture containing one or more liquids, nutrients, andthe like introduced to the loop reactor 100 by a nutrient and/or mineralsupply subsystem. In at least some instances, the liquid effluent may beremoved from the loop reactor and returned to the gas/liquid separationvessel 109 by spraying onto the surface of the multi-phase mixture inthe gas/liquid separation vessel 109 in order to reduce foaming withingas/liquid separation vessel 109. Anti-foam agents may be added to theliquid effluent sprayed into gas/liquid separation vessel 109 or maybesprayed into gas/liquid separation vessel 109 without the liquideffluent. In at least some instances, the liquid effluent may includeone or more useful compounds. For example, the liquid effluent maycontain an amount of one or more liquid C₂₊ hydrocarbon compoundsincluding, but not, limited to alcohols, ketones, glycols, and othercompounds based thereupon having value as either a finished product oras a raw material in a subsequent process. Such useful hydrocarboncompounds may be separated from the liquid effluent.

In some instances, systems for stimulating the production of biomass inaccordance with embodiments described herein are used to produce naturalor non-natural products, such as ethanol, acetate, butanol, isoprene,propylene, isoprene, enzymes, or other metabolites or cellular productswherein the product is derived from a microorganism. In such cases, theproducts may be present in either the gas phase or liquid phaseseparated in gas/liquid separation vessel 109 depending on the physicalproperties of the product.

As illustrated in FIG. 1, the outlet of fluid moving device 107 is influid communication with an inlet of substantially horizontal zone 104of loop reactor 100. Substantially horizontal zone 104 of loop reactor100 extends from its inlet to its outlet. The outlet of horizontal zone104 is in fluid communication with the inlet to substantially verticalupflow zone 105. Substantially horizontal zone 104 can be formed frompiping made from materials that do not adversely affectreaction/fermentation processes carried out using loop reactor 100. Forexample, substantially horizontal zone 104 can be formed from pipingmade from the materials described above for elements of loop reactor100. The cross-sectional area of substantially horizontal zone 104 maybe constant or substantially horizontal zone 104 may include one or moresections that have different cross-sectional areas. Reference to thecross-sectional area of substantially horizontal zone 104 in the presentdisclosure does not include the cross-sectional area of gas/liquidseparation vessel 109. The inner diameter of substantially horizontalzone 104 may vary, with exemplary diameters ranging from about 20centimeters to 3 meters. Other exemplary diameters range from 25centimeters to 2.5 meters. When substantially horizontal zone 104includes sections of differing cross-sectional areas, the sections ofsubstantially horizontal zone 104 having larger cross-sectional areahave cross-sectional areas that are at most three times thecross-sectional area of the sections of substantially horizontal zone104 having smaller cross-sectional areas. In other exemplaryembodiments, sections of substantially horizontal zone 104 having largercross-sectional area, have cross-sectional areas that are at most twotimes the cross-sectional area of the sections of substantiallyhorizontal zone 104 having smaller cross-sectional areas. In yet otherexemplary embodiments, sections of substantially horizontal zone 104having larger cross-sectional area, have cross-sectional areas that areat most 0.5 times the cross-sectional area of sections of substantiallyhorizontal zone 104 having smaller cross-sectional areas. The length ofsubstantially horizontal zone 104 can vary depending upon a number offactors, including the desired length of time the multi-phase mixtureresides in substantially horizontal zone 104. The length ofsubstantially horizontal zone 104 may also be determined based on otherfactors such as, but not limited to total reactor/liquid volume desired,total pressure drop across substantially horizontal zone 104, desiredsubstrate utilization and yield. In exemplary embodiments, substantiallyhorizontal zone 104 can vary in the length at its centerline from about30 m to about 250 m, 40 m to about 200 m, 50 m to about 150 m and 60 toabout 100 m.

The embodiment of substantially horizontal zone 104 illustrated in FIG.1 is U-shaped, including two elbows 137 that bend at 90° angles whenviewed from above. Substantially horizontal zone 104 can take othershapes. For example, substantially horizontal zone 104 can include morethan the two 90° elbows 137 or it can include more than one elbow thatis less than 90°. In other embodiments, substantially horizontal zone104 can include numerous elbows that are greater than 90° or less than90°.

Outlet of substantially horizontal zone 104 may be elevated relative tothe inlet of substantially horizontal zone 104. Substantially horizontalzone 104 may accommodate for this difference in elevation between itsinlet and its outlet by being sloped. The specific slope ofsubstantially horizontal zone 104 or portions of substantiallyhorizontal zone 104 depend in part on the length of substantiallyhorizontal zone 104, the vertical distance between the centerline ofsubstantially horizontal zone 104 at its inlet and the centerline ofsubstantially horizontal zone 104 at its outlet. Substantiallyhorizontal zone 104 can be sloped upward from its inlet to its outlet toaccommodate for the change in elevation between its inlet and itsoutlet. Alternatively, a portion of substantially horizontal zone 104can be sloped downward and a portion of substantially horizontal zone104 can be sloped upward. In such alternative embodiments, the portionof substantially horizontal zone 104 that is sloped upward accounts forthe loss in elevation resulting from the presence of the downward slopedportion of substantially horizontal zone 104 and the difference inelevation between the inlet of substantially horizontal zone 104 and theoutlet of substantially horizontal zone 104. For example, the portion ofsubstantially horizontal zone 104 extending from its inlet to the first90° elbow 137 in FIG. 1 can be sloped downward, and the portion ofsubstantially horizontal zone 104 extending from the first or secondelbow 137 can be sloped upward to the outlet of substantially horizontalzone 104.

Exemplary embodiments illustrated in FIG. 1 include a plurality ofstatic mixers, positioned along the length of substantially horizontalzone 104. Benefits of the use of static mixers are described in U.S.Pat. No. 7,579,163 and include mixing of the nutrient gases into themulti-phase mixture. Exemplary types of static mixers are also describedin the '163 patent. Static mixers that can be used in embodimentsdescribed are not limited to those described in the '163 patent. Staticmixers other than those described in the '163 patent can be used in theembodiments described herein. For example, other types of static mixersare available from companies such as StaMixCo LLC of Brooklyn, N.Y. andSulzer Management Ltd. of Winterthur, Switzerland.

In exemplary embodiments, system 200 includes a nutrient and/or mineralsupply subsystem for introducing nutrients and minerals intosubstantially horizontal zone 104 at one or more locations. Suchnutrients include nutrients capable of supporting or transportingdissolved or suspended sustenance to biomass forming microbiologicalorganisms in the multi-phase mixture within the loop reactor 100. Inexemplary embodiments, nutrients and minerals may be introduced at oneor more locations along substantially horizontal zone 104. The nutrientsupply subsystem may also provide gaseous substrates/nutrients forintroduction into a liquid culture medium to form a multi-phase mixtureof the liquid culture medium and supplied gaseous substrates/nutrients.Such gaseous substrates/nutrients can include a single gas or acombination of gases capable of supporting or providing sustenance ornutrients to the biomass producing biological organisms in the loopreactor 100. Exemplary nutrients include natural gas, nitrogen, oxygenand ammonia water. A source of steam can be provided for thermal energyand cleaning purposes. Nutrients that can be supplied by nutrientsubsystem are not limited to natural gas, nitrogen, oxygen and ammoniumwater. Other nutrients/minerals, such as methane, syngas, water,phosphate (e.g., as phosphoric acid), nitrates, urea, magnesium,calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt andmolybdenum, typically used as sulfates, chlorides or nitrates can alsobe provided by the nutrient subsystem.

In exemplary embodiments, system 100 may include a heat transfer unitoperation for introducing or removing thermal energy from themulti-phase mixture in loop reactor 100. The heat transfer unitoperation can introduce thermal energy to or remove thermal energy fromthe multi-phase mixture in loop reactor 100 at one or more locations. Inat least some instances, the microbiological activity that occurs withinthe loop reactor 100 generates heat as a byproduct. Left uncontrolled,such heat can adversely affect the metabolism or health of themicrobiological organisms within the loop reactor 100. Alternatively,microbiological organisms may also have a temperature below which themetabolism or health of the organism is adversely affected. As such, thebiological organisms within the loop reactor 100 have a definedtemperature range providing optimal growth and metabolic conditions. Inat least some instances, the multi-phase mixture within the loop reactor100 can be maintained at a temperature of about 130° F. or less; about120° F. or less; about 110° F. or less; about 100° F. or less; about 95°F. or less; about 90° F. or less; about 85° F. or less; or about 80° F.or less using the heat transfer unit operation. In at least someinstances, the multi-phase mixture within the loop reactor 100 can bemaintained at a temperature of from about 55° F. to about 120° F.; about60° F. to about 110° F.; about 110° F. to about 120° F.; about 100° F.to about 120° F.; about 65° F. to about 100° F.; about 65° F. to about95° F.; or about 70° F. to about 90° F. using heat transfer unitoperation.

Gas/liquid separation vessel 109, substantially vertical upflow zone 105and/or substantially horizontal zone 104 of loop reactor 100 may includea desorption gas inlet. Desorption gas inlet is in fluid communicationwith a source of desorption gas, e.g., nitrogen, and in fluidcommunication with gas/liquid separation vessel 109, substantiallyvertical upflow zone 105 and/or substantially horizontal zone 104 ofloop reactor 100. Thus, in accordance with embodiments of loop reactorsin accordance with embodiments described herein, desorption gas can beintroduced into gas/liquid separation vessel 109, substantially verticalupflow zone 105 and/or substantially horizontal zone 104 of loop reactor100. Introducing a desorption gas into the multi-phase mixture causes adecrease in the partial pressure of other gases present in themulti-phase mixture (e.g., carbon dioxide and methane). Reducing thepartial pressure of other gases present in the multi-phase mixture canhave the effect of reducing the mass transfer of nutrient gases into themicroorganism and/or causing the other gases to come out of solution.

FIG. 10 shows a high level method of operation 500 of a system 200 forstimulating production of biomass using one or more loop reactors 100described in detail above with regard to FIGS. 1-9. Such systemsadvantageously introduce one or more gaseous substrates and a liquidmedia containing one or more nutrients into a liquid culture mediacontaining at least one microorganism capable of utilizing the gaseoussubstrates and liquid nutrients to grow. The combination of the one ormore gaseous substrates, liquid media containing one or more nutrientsand liquid culture media containing at least one microorganism resultsin a multi-phase mixture that is circulated through loop reactor 100.The conditions within loop reactor 100 promote mass transfer andsubsequent microbiological uptake of the gaseous substrate and liquidnutrients, reduction of pressure within the loop reactor and desorptionof gases from the multi-phase mixture. The multi-phase mixture afterpassing through the substantially horizontal zone 104 and thesubstantially vertical upflow zone 105 of loop reactor 100 is receivedby a gas/liquid separation unit operation 109 where the multi-phasemixture is separated into liquid and gas phases. The method commences at502.

At 504 a gaseous substrate is dispersed within the liquid media to formthe multi-phase mixture. Such dispersion may occur downstream of fluidmoving device 107 and upstream of gas/liquid separation vessel 109. Insome instances, gaseous substrate may be dispersed at multiple pointsalong loop reactor 100 and the gaseous substrate at each dispersionpoint may have the same or a different temperature, pressure,composition, or combinations thereof. The ability to vary physical orcompositional properties of the gaseous substrate at different locationsalong the loop reactor 100 advantageously permits the tailoring of thegaseous substrate not only to the specific microbiological speciespresent in the multi-phase mixture, but also to the specific location ofthe microbiological species within loop reactor 100 based on thedispersion point of the gaseous substrate.

At 506 the multi-phase mixture is flowed through loop reactor 100. Asthe multi-phase mixture flows through the loop reactor 100, it maycontact a plurality of static mixers located within loop reactor 100,which promote the mixing of the gaseous substrate and/or nutrients intothe liquid culture medium. By adjusting or otherwise controlling theflow rate of the multi-phase mixture through loop reactor 100, thelength of time the bubbles of gaseous substrate and nutrients are incontact with the microorganism(s) can be modified. Increasing the lengthof time the bubbles of gaseous substrate and nutrients are in contactwith the microorganism(s) can increase the amount of mass transfer ofgaseous materials into the microorganisms and the microbiological uptakeof gaseous materials by the microorganism. Conversely, decreasing thelength of time the bubbles of gaseous substrate and nutrients are incontact with the microorganism(s) can decrease the amount of masstransfer of gaseous materials into the microorganisms and themicrobiological uptake of gaseous materials by the microorganisms. Insome instances, the length of time the bubbles of the gaseous substrateand nutrients are in contact with the microorganisms can be measured andcontrolled. For example, control subsystem 290 in FIG. 2 can alter,adjust or control the fluid velocity of the multi-phase mixture throughloop reactor 100. In some instances, the temperature, pressure, orcomposition of the gaseous substrate may be altered, adjusted orcontrolled via control subsystem 290 to maintain a desired gas substratebubble size within loop reactor 100. In other instances, thetemperature, pressure, or composition of the gas substrate may bealtered, adjusted or controlled via control subsystem 290 to maintainthe concentration of one or more gas substrate components (e.g.,methane, carbon dioxide, hydrogen, oxygen, nitrogen, etc.) within theliquid phase of the multi-phase mixture.

At 508 the temperature of the multi-phase mixture within loop reactor100 can be altered, adjusted, or controlled to maintain the temperaturewithin a defined temperature range. In at least some instances, thedefined temperature range may be selected or otherwise chosen based atleast in part on the microbiological species used within system 100.Excess heat may be generated as a byproduct by the microbiologicalorganisms responsible for at least a portion of the activity withinsystem 200. This excess heat, if left uncontrolled, could inhibit oradversely affect the growth or metabolism of some or all of themicrobiological organisms within system 200. In at least some instances,cooling of the multi-phase mixture in loop reactor 100 may be providedto maintain the temperature of the multi-phase mixture in loop reactor100 within a defined range. Such cooling may include passage of acooling media through reservoirs or coils thermally conductively coupledto the loop reactor 100 or a conduit which has diverted a portion of themulti-phase mixture out of the loop reactor 100 to a heat transfer unitoperation. In at least some instances, control subsystem 290 may controlthe flow rate or temperature of the cooling media passed through thereservoirs or coils that are thermally conductively coupled to loopreactor 100 or a conduit which has diverted portion of the multi-phasemixture out of loop reactor 100 to a heat transfer unit operation. Inother instances, the heat produced by the microbiological species may beinsufficient to maintain the multi-phase mixture in loop reactor 100within a desired temperature range. Such may occur, for example, inextremely cold environments where loop reactor 100 is located in anexposed or partially exposed exterior location. In some instances, thereservoirs or coils thermally conductively coupled to loop reactor 100or the conduit which has diverted portion of the multi-phase mixture outof loop reactor 100 to a heat transfer unit operation may be used towarm the multi-phase mixture. In at least some instances, controlsubsystem 290 may control the flow rate or temperature of the warmingmedia passed through the reservoirs or coils that are thermallyconductively coupled to the loop reactor 100 or the conduit which hasdiverted portion of the multi-phase mixture out of the loop reactor 100to a heat transfer unit operation.

At 510, the pressure on the gas substrate bubbles traveling with themulti-phase mixture through loop reactor 100 is decreased by flowing themulti-phase mixture through a substantially vertical upflow zone 105.The pressure decrease at 510 can, in some instances, advantageouslyincrease the rate at which gas substrate bubbles and other gases desorbfrom the multi-phase mixture.

At 512, the multi-phase mixture exits substantially vertical upflow zone105 and flows to the gas/liquid separation vessel 109. Gaseous materialthat has desorbed from the multi-phase mixture can also flow to thegas/liquid separation vessel 102 along with the multi-phase mixture. Themulti-phase mixture entering the gas/liquid separation vessel 109 caninclude, but is not limited to the liquid containing unabsorbednutrients, microorganisms and gas substrate bubbles containingundissolved and unabsorbed gas substrate. Gas and liquid enteringgas/liquid separation vessel 109 separate into a gas phase and a liquidphase within gas/liquid separation vessel 109. Gases can be collectedfrom the headspace of gas/liquid separation vessel 109 while liquid canbe removed from the bottom of gas/liquid separation vessel 109. Inaddition to liquid, microorganisms can also be collected in gas/liquidseparation vessel 109 and removed from the bottom thereof at step 514.The liquid and microorganisms removed from the bottom of gas/liquidseparation vessel 109 can be delivered to the inlet of fluid movingdevice 107 via substantially vertical down flow zone 103 forrecirculation through loop reactor 100. In at least some instances, atleast a portion of the collected gas may be removed from gas/liquidseparation vessel 109 and subsequently processed or separated. At leasta portion of the collected gas may be recycled to the loop reactor as agas substrate. In some instances, at least a portion of the collectedgas may be sold or otherwise disposed of. In at least some instances, atleast a portion of the collected gas may be sold or traded as a fungiblecommodity. In at least some instances, the collected gas may include oneor more C₂₊ hydrocarbon gases and compounds based thereupon having valueas either a finished product or as a raw material in a subsequentprocess. In some instances, the reactor is used to produce natural ornon-natural products, such as ethanol, acetate, butanol, isoprene,propylene, farnesene, enzymes, or other metabolites or cellular productswherein the product is derived from a microorganism. In such cases, theproducts may be present in either the gas effluent or the liquideffluent depending on the physical properties of the product.

In at least some instances, at least a portion of the collected liquidmay be subsequently processed or separated. For example, at least aportion of the liquid separated from the multi-phase mixture, which mayor may not include biosolids, can be recycled through loop reactor 100.For example, at least a portion of the separated liquid containingbiosolids may be combined with additional liquids and flowed through theloop reactor 100. Such recycle may advantageously provide an ongoing,continuous or semi-continuous, inoculation of the loop reactor 100 withestablished biological species. In some instances, at least a portion ofthe separated liquid may be collected and sold or otherwise disposed of.In at least some instances, at least a portion of the separated liquidmay be sold or traded as a fungible commodity. In at least someinstances, the separated liquid may include one or more C₂₊ hydrocarbonliquids, including but not limited to one or more alcohols, glycols, orketones.

Referring to FIG. 11, in additional embodiments of the subject matterdescribed herein, gas/liquid separation vessel 109 may include one ormore flow guides 401 which cause flow of the multi-phase mixture withinintermediate section 301 and inlet end section 305 of gas/liquidseparation vessel 109 to occur with less channeling, less turbulenceand/or less speed. Reducing channeling of the flow of the multi-phasemixture in intermediate section 301 and inlet end section 305, reducingturbulence of the flow of the multi-phase mixture in intermediatesection 301 and inlet end section 305 and/or increasing residence timeof the multi-phase mixture within intermediate section 301 and inlet endsection 305 can result in an increase in the amount of gas that desorbsfrom the multi-phase mixture within gas/liquid separation vessel 109.One example of suitable flow guides 401 is illustrated in FIGS. 4 and 11and includes a v-shaped vertical baffle 401 located within intermediatesection 301 and inlet end section 305. Another example of a suitableflow guide includes two or more parallel vertical baffles located withinintermediate section 301 and inlet end section 305. Such parallelvertical baffles have their lengths parallel with the direction of bulkflow of the multi-phase mixture through gas/liquid separation vessel109.

The above description of illustrated embodiments, including what isdescribed in the Abstract, is not intended to be exhaustive or to limitthe embodiments to the precise forms disclosed. Although specificembodiments of and examples are described herein for illustrativepurposes, various equivalent modifications can be made without departingfrom the spirit and scope of the disclosure, as will be recognized bythose skilled in the relevant art. The teachings provided herein of thevarious embodiments can be applied to other systems for stimulating theproduction of biomass, fermentors and fermentation systems. Such systemsfor stimulating the production of biomass, fermentors and fermentationsystems may include loop reactors or fermentors for purposes other thanchemical intermediate production, and may include loop reactors,fermentors and fermentation systems useful in food or beverageproduction. Similarly, the ancillary systems described herein, includingthe fluid moving device, nutrient and/or mineral supply subsystem, heattransfer unit operation and the control subsystem may include a singlesystem, for example a package heat exchanger or package control system,or may include a custom designed subsystem including any number ofsubcomponents that are physically, fluidly, and communicably coupled ina manner facilitating the controlled production and distribution ofcooling or warming media (i.e., by the heat transfer unit operation).The control subsystem can include an integrated or distributed controlsystem that provides monitoring, alarming, control, and control outputfor all or a portion of the biomass production system or any of theancillary subsystems. The control subsystem may also include any numberof individual loop controllers and the like for control of one or moreaspects of the biomass production system or any of the ancillarysubsystems.

The foregoing detailed description has set forth various embodiments ofthe devices and/or processes via the use of process flow diagrams andexample methods. Insofar as such block diagrams, schematics, andexamples contain one or more functions and/or operations, it will beunderstood by those skilled in the art that each function and/oroperation within such block diagrams, flowcharts, or examples can beimplemented, individually and/or collectively, using wide range ofoff-the-shelf or customized components that are well known to those ofskill in the chemical engineering arts. The microbiological specieslisted herein are intended to provide a sample of the potentialmicrobiological species that can be supported in a system for promotingthe production of biomass and loop reactors as described herein.

The various embodiments described above can be combined to providefurther embodiments. These and other changes can be made to theembodiments in light of the above-detailed description. In general, inthe following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A process for stimulating production of biomass comprising: flowingthrough a loop section of a loop reactor, a multi-phase mixture of a gasand a liquid culture medium; introducing a nutrient into the multi-phasemixture; separating the multi-phase mixture of a gas and a liquidculture medium into a gas phase and a liquid phase in a gas/liquidseparation vessel by: flowing the multi-phase mixture of a gas and aliquid culture medium into a lower portion of the gas/liquid separationvessel through an inlet side section of the gas/liquid separationvessel, the inlet side section of the gas/liquid separation vesselhaving a shape of an oblique conical frustum and having an increasingdiameter in a direction of flow of the multi-phase mixture through theinlet side section; and flowing the multi-phase mixture through anintermediate section located between an outlet side section of thegas/liquid separation vessel and the inlet side section of thegas/liquid separation vessel, the intermediate section having a constantdiameter; and removing the liquid phase from an outlet of the gas/liquidseparation vessel and delivering the removed liquid phase to an inlet ofthe loop section.
 2. The process of claim 1, wherein delivering theremoved liquid phase to the inlet of the loop section includes flowingthe removed liquid phase through a conduit of non-increasing diameter.3. The process of claim 1, wherein separating the multi-phase mixture ofa gas and a liquid culture medium into a gas phase and a liquid phase inthe gas/liquid separation vessel further comprises flowing themulti-phase mixture through the inlet side section and the intermediatesection of the gas/liquid separation vessel, wherein the inlet sidesection and the intermediate section include a lowermost edge thatcontacts a common plane.
 4. The process of claim 1, wherein thenutrients include a C₁ compound.
 5. The process of claim 4, wherein theC₁ compound is methane.
 6. The process of claim 1, further comprisingflowing the multi-phase mixture of a gas and a liquid culture mediumthrough a substantially vertical downflow zone before flowing themulti-phase mixture through the loop section of a loop reactor.
 7. Theprocess of claim 1, further comprising flowing the multi-phase mixtureof a gas and a liquid culture medium through a substantially verticalupflow zone after flowing the multi-phase mixture through the loopsection of a loop reactor.
 8. The process of claim 1, wherein alowermost edge of the intermediate section, the outlet side section andthe inlet side section of the gas/liquid separation vessel contact acommon plane.
 9. The process of claim 1, wherein flowing the multi-phasemixture of a gas and a liquid culture medium into a lower portion of thegas/liquid separation vessel through an inlet side section of thegas/liquid separation vessel having a shape of an oblique conicalfrustrum includes flowing the multi-phase mixture of a gas and a liquidculture medium through an inlet side section of the gas/liquidseparation vessel having a shape of an oblique conical frustrumincluding a cone angle that ranges between 5 to 30°.
 10. The process ofclaim 1, wherein the gas/liquid separation vessel has a longitudinalaxis and the inlet side section has a length L_(a) measured along thelongitudinal axis of the gas/liquid separation vessel that is greaterthan a length L_(i) of the intermediate section measured along thelongitudinal axis of the gas/liquid separation vessel.
 11. A process forstimulating production of biomass comprising: flowing through asubstantially horizontal section of a loop reactor, a multi-phasemixture of a gas and a liquid culture medium; introducing a nutrientinto the multi-phase mixture; separating the multi-phase mixture of agas and a liquid culture medium into a gas phase and a liquid phase in agas/liquid separation vessel by: flowing the multi-phase mixture of agas and a liquid culture medium into a lower portion of the gas/liquidseparation vessel through an inlet side section of the gas/liquidseparation vessel, the inlet side section of the gas/liquid separationvessel having a shape of an oblique conical frustum and having anincreasing diameter in a direction of flow of the multi-phase mixturethrough the inlet side section; and flowing the multi-phase mixturethrough an intermediate section located between an outlet side sectionof the gas/liquid separation vessel and the inlet side section of thegas/liquid separation vessel, the intermediate section having a constantdiameter; and removing the liquid phase from an outlet of the gas/liquidseparation vessel and delivering the removed liquid phase to an inlet ofthe substantially horizontal section.
 12. The process of claim 11,wherein delivering the removed liquid phase to the inlet of the loopsection includes flowing the removed liquid phase through a conduit ofnon-increasing diameter.
 13. The process of claim 11, wherein flowingthe multi-phase mixture of a gas and a liquid culture medium into alower portion of the gas/liquid separation vessel through an inlet sidesection of the gas/liquid separation vessel having a shape of an obliqueconical frustrum includes flowing the multi-phase mixture of a gas and aliquid culture medium through an inlet side section of the gas liquidseparation vessel having a shape of an oblique conical frustrumincluding a cone angle that ranges between 5 to 30°.
 14. The process ofclaim 11, wherein the gas/liquid separation vessel has a longitudinalaxis and the inlet side section has a length L_(a) measured along thelongitudinal axis of the gas/liquid separation vessel that is greaterthan a length L_(i) of the intermediate section measured along thelongitudinal axis of the gas/liquid separation vessel.
 15. The processof claim 14, wherein the ratio of L_(a)/L_(i) is between 0.08 to 0.2.16. The process of claim 14, wherein length L_(i) of the intermediatesection is between 2 to 8 meters.
 17. The process of claim 11, whereinthe flowing the multi-phase mixture through an intermediate sectionincludes flowing the multi-phase mixture through an intermediate sectionthrough an intermediate section that has a diameter D that is between 2to 8 meters.
 18. The process of claim 11, wherein the flowing themulti-phase mixture through an intermediate section includes flowing themulti-phase mixture through an intermediate section of a plurality ofgas/liquid separation vessels, the plurality of gas/liquid separationvessels arranged in series or arranged in parallel.
 19. The process ofclaim 11, further comprising flowing the multi-phase mixture of a gasand a liquid culture medium through a substantially vertical upflow zoneafter flowing the multi-phase mixture through the substantiallyhorizontal section of a loop reactor and flowing the multi-phase mixtureof a gas and a liquid culture medium from the substantially verticalupflow zone into the inlet side section via a bend having a radius ofcurvature between 0.5 to 3 times a diameter of the vertical upflow zone.20. The process of claim 11, wherein delivering the removed liquid phaseto the inlet of the substantially horizontal section includes flowingthe removed liquid phase through a conduit of non-increasing diameter.